Alcohols, C12-15, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Oral (rat, m/f, OECD 422): NOAEL (reproduction) = 1000 mg/kg bw/day

Oral (rat, m/f, OECD 422): NOAEL (systemic toxicity) = 1000 mg/kg bw/day

Oral (rat, m/f, OECD 422): NOAEL (local toxicity) = 1000 mg/kg bw/day

 

Conclusion based on data obtained with alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) and considering all the available data on toxicity to reproduction in the Alcohol Ethoxylates (AE) category, in a Weight-of-Evidence approach.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 MAR 2020 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females: nulliparous and non-pregnant
- Age at study initiation: males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: males 266-317 g, females 201-271 g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 46-56
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES
From: 18 MAR 2020 to 20 MAY 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until dosing.
- Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure (Test Facility Study No. 20219687).
- Concentration in vehicle: 4 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Details on mating procedure:

Mating procedure
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in
the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure (Test Facility Study No. 20219687).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was less than or equal to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-64 days.
Females which failed to deliver or had a total litter loss were treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finding Study with oral gavage administration of the test material in rats (Test Facility Reference No. 20219688), and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

BODY WEIGHT: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

CLINICAL CHEMISTRY AND HAEMATOLOGY: Blood of all F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthasia (isoflurane). F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

SERUM HORMONES: Measurement of total T4 was conducted for F0-males.

NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor activity.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

GROSS EXAMINATION OF DEAD PUPS
Pups found dead were examined for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the last litter of each generation was weaned.

GROSS PATHOLOGY AND ORGAN WEIGHTS: All animals were subjected to a full post mortem examination and the following organs weighed; Brain, cervix, epididymis, adrenal, coagulation gland, parathyroid, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus

HISTOPATHOLOGY: Histopathology was conducted for the following tissues: Aorta, nasopharynx, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal, coagulation gland, harderian, lacrimal, mammary, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid, gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea urinary bladder uterus, vagina.

Postmortem examinations (offspring):

SACRIFICE
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two or three pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
-Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females/Number of females mated x 100

Offspring viability indices:

For each group, the following calculations were performed. Group mean values of duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

Gestation index (%):Number of females with living pups on Day 1/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%):Total number of offspring born/Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/
Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among all animals of the 300 and 1000 mg/kg/day dose groups and some animals of the 100 mg/kg/day dose group was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Incidental findings that were noted included rales, alopecia and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item up to 1000 mg/kg/day.
One female of the control group and one female at 300 mg/kg/day were euthanized on Lactation Days 7 and 3, respectively, as they had a total litter loss.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered not affected by treatment with the test item up to 1000 mg/kg/day.
At 1000 mg/kg/day, mean relative food consumption was increased (1.27x of control; statistically significant) in females over lactation Days 1-4. No toxicologically relevance was attached to this finding, as food intake had returned to control levels in the subsequent intervals.
Any statistically significant changes in food consumption before or after correction for body weight observed in Groups 2 and 3 were considered to be unrelated to treatment since no trend was apparent regarding dose.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
The statistically significant increase in mean activated partial thromboplastin time (APTT) in males at 100 and 300 mg/kg/day was considered unrelated to administration of the test item due to absence of a dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered affected by treatment with the test item at 300 and 1000 mg/kg/day.
The following statistically significant changes distinguished treated from control animals:
• Mean albumin concentration was slightly increased (both 1.06x; above historical control range) in males at 300 and 1000 mg/kg/day.
• Mean urea concentration was increased (1.37x; within historical control range) in males at 1000 mg/kg/day.
• Mean cholesterol concentration was decreased (0.74 and 0.65x, respectively; both below historical control range) in males at 300 and 1000 mg/kg/day.
• Mean bile acid concentration was increased (2.90x; within historical control range) in females at 1000 mg/kg/day.
These changes were considered as non-adverse since they were not associated with any adverse pathological alterations.

Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.

Endocrine findings:

not specified

Description (incidence and severity):

Serum levels of total T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional tests were inadvertently not performed in females.
Functional observation parameters were considered not affected by treatment with the test item up to 1000 mg/kg/day in males.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined males up to 1000 mg/kg/day. Grip strength was similar between control and high dose males.
Motor activity was similar between treated and control male groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver, jejunum and forestomach of males and females at 1000 mg/kg/day, in the thyroid gland of males starting at 100 mg/kg/day and in the adrenal gland of females at 1000 mg/kg/day. These changes are summarized in Table 2, 3 and 4.
Liver: Centrilobular hepatocellular hypertrophy was noted at 1000 mg/kg/day in all males up to slight degree and in 2/6 females at minimal degree.
Jejunum: Vacuolation in the lamina propria was noted at 1000 mg/kg/day in 4/5 males up to slight degree and in 3/5 females at minimal degree.
Forestomach: Minimal squamous cell hypertrophy was noted at 1000 mg/kg/day in 4/5 males and in a single female.
Thyroid glands: An increased incidence and/or severity of follicular cell hypertrophy was noted starting at 100 mg/kg/day in males up to slight degree.
Adrenal glands: Diffuse cortical hypertrophy was noted at 1000 mg/kg/day in 3/5 females up to slight degree.
All of these changes were considered non-adverse.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
All females had regular cycles of 4 to 5 days. Extend di-estrous occurred during the mating in a number of females at 300 mg/kg/day with a regular cycle during premating. One female at 300 mg/kg/day had an inconclusive cycle determination during the pre-mating phase. Given the absence of a dose-related incidence, this finding did not indicate a relation with treatment with the test item.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed.
One male from the 1000 mg/kg/day treatment group showed moderate degeneration/atrophy of the germ cells and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This animal had no normal spermatogenic staging profile.
In all other males the testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 1/10 couples of the control group , 1/10 couples of the 100 mg/kg/day group and 1/10 couples of the 1000 mg/kg/day group with no offspring. A single female of the control group and of the 300 mg/kg/day group had a total litter loss.
One male from the 1000 mg/kg/day treatment group showed moderate degeneration/atrophy of the germ cells and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This animal had no normal spermatogenic staging profile.
For the other couples, no abnormalities were seen in the reproductive organs, which could account for their lack of offspring or total litter loss.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
Mating index, precoital time, number of implantation sites, fertility index and gestation index was considered not to be affected by treatment with the test item up to 1000 mg/kg/day. No signs of difficult or prolonged parturition and no deficiencies in maternal care were noted among the pregnant females.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 1000 mg/kg/day.
For the pups of two females in the control group and one female at 300 mg/kg/day that were found dead at PND 1, missing on PND 2 or 3, or euthanized in extremis on PND 7, absence of milk in the stomach were noted on PND 1, 2, 3 or 7. In addition, for the pups of a control female and one female at 300 mg/kg/day that were missing on PND 3 or euthanized in extremis on PND 7, a dehydrated appearance was noted on PND 1 and 7.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant.

Description (incidence and severity):

Pups of two females in the control group and one female at 300 mg/kg/day were found dead at PND 1, missing on PND 2 or 3, or euthanized in extremis on PND 7. In addition, pups of a control female and one female at 300 mg/kg/day were missing on PND 3 or euthanized in extremis on PND 7.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered to be affected by treatment with the test item at 1000 mg/kg/day.
At PND 7 and 13, mean body weights of male pups were statistically significantly decreased (0.88 and 0.86x of control, respectively) at 1000 mg/kg/day. In addition, mean body weights of female pups at 1000 mg/kg/day were decreased (0.90x of control; not statistically significant) at PND 13. At PND 13, male and female pup weights of 3/9 litters at 1000 mg/kg/day were below the historical control range.
In addition, the body weight of one pup in two litters at 100 mg/kg/day were significantly lower compared to the control mean at PND 7 and 13 indicating a growth retardation. In the absence of a dose-related trend of the same order of magnitude, these single occurrences in both litters were not considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum levels of total T4 in male and female PND 14-16 pups were affected by treatment with the test item at 100, 300 and 1000 mg/kg/day. However, under the conditions of this screening study no adverse effect was observed that could be linked to the increase of total T4 and therefore this increase was not taken into account when determining the developmental NOAEL.
At 100, 300 and 1000 mg/kg/day, mean levels of total T4 were increased to 1.28, 1.18 and 1.19x of control, respectively, in male PND 14-16 pups (statistically significant at 100 mg/kg/day only) and to 1.42, 1.26 and 1.33x of control, respectively, in female PND 14- 16 pups (statistically significant in all treatment groups). The mean values remained within the historical control range.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1. Mean Percent Organ Weight Differences from Control Group

		Males			Females
Dose level (mg/kg/day):	100	300	1000	100	300	1000
LIVER
Absolute	14*	18**	33**	0	27**	39**
Relative to body weight	8*	14**	36**	3	20**	36**
THYROID GLANDS
Absolute	5	2	15	na	na	na
Relative to body weight	2	0	18*	na	na	na
ADRENAL GLANDS
Absolute	na	na	na	4	16	35**
Relative to body weight	na	na	na	13	17	39**

na = not applicable

*: P<0.05, **: P<0.01

Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
LIVER ^a	5	5	5	6	6	5	6	6
Centrilobular hypertrophy
Minimal	-	-	-	1	-	-	-	2
Slight	-	-	-	5	-	-	-	-
JEJUNUM ^a	5	5	5	5	5	5	5	5
Vacuolation
Minimal	-	-	-	3	-	-	-	3
Slight	-	-	-	1	-	-	-	-
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia
Minimal	-	-	-	4	-	-	-	1

^a = Number of tissues examined from each group.

Table 3. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males
Dose level (mg/kg/day):	0	100	300	1000
THYROID GLANDS ^a	5	5	5	5
Follicular cell hypertrophy
Minimal	-	3	3	4
Slight	-	-	1	1

^a = Number of tissues examined from each group.

Table 4. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Females
Dose level (mg/kg/day):	0	100	300	1000
ADRENAL GLANDS ^a	5	5	6	5
Diffuse cortical hypertrophy
Minimal	-	-	-	1
Slight	-	-	-	2

^a = Number of tissues examined from each group.

 

 

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Alcohols, C12-15, branched and linear, ethoxylated

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies:
With respect to reproductive toxicity, only a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test according to OECD guideline 422 is available with the registered substance. In this study, no relevant effects in relation to reproductive and developmental toxicity have been observed. In addition, a subchronic (90-day) oral repeated dose toxicity study, a prenatal developmental toxicity study in rats and a prenatal developmental toxicity study in rabbits, all conducted with the registered substance, did not reveal any observations in reproductive tissues and organs indicative of reproductive / developmental toxicity.
- Available non-GLP studies:
No study to meet the information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 with the registered substance is available.
- Historical human data:
The registrant is not aware of any relevant human data suitable to satisfy the information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 with the registered substance.
- (Q)SAR:
No QSARs are available to reliably predict the parameters assessed in an extended one-generation reproductive toxicity study (OECD 443). Therefore, a QSAR assessment is not possible.
- In vitro methods:
No in vitro methods are available to reliably predict the parameters assessed in an extended one-generation reproductive toxicity study (OECD 443). Therefore, in vitro studies are not possible.
- Weight of evidence:
No adequate and reliable data are available that possibly could be used for a Weight-of-Evidence assessment of reproductive toxicity for the registered substance.
- Grouping and read-across:
The registered substance is a member of the Alcohol Ethoxylates (AE) category. The category contains structurally similar substances sharing a similar toxicokinetic behaviour and similar toxicological properties. In fact, robust read-across approaches are applied for several human health-related endpoints, including systemic and local toxicity. However, only four of the AE substances are subject to information requirements according to Annex X of the REACH Regulation (EC) No. 1907/2006 and no adequate and reliable studies concerning reproductive toxicity (other than screening studies) are available in the database of the category. It is intended to perform extended one-generation reproductive toxicity studies (OECD 443) for two of the four substances and to apply a read-across approach to cover the information requirement of Annex X, Section 8.7.3. for the remaining two substances. Based on the structural properties of the registered substance, it has been selected as one of the source substances. In consequence, the only way to comply with the standard information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 is to perform an extended one-generation reproductive toxicity study according to OECD guideline 443 with the registered substance.
- Substance-tailored exposure driven testing:
The registered substance is used in a variety of applications. None of the conditions and prerequisites of Annex XI, Section 3. of the REACH Regulation (EC) No. 1907/2006 can be met. Therefore, substance-tailored exposure driven testing cannot be applied for the registered substance.
- Approaches in addition to above:
Not applicable
- Other reasons:
Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Annex X, Section 8.7. (Reproductive Toxicity), Column 2, of the REACH Regulation (EC) No. 1907/2006 stipulates the following specific adaption options:
‘The studies need not be conducted if:
• the substance is known to be a genotoxic carcinogen, meeting the criteria for classification both in the hazard class germ cell mutagenicity (category 1A or 1B or 2) and carcinogenicity (category 1A or 1B), and appropriate risk management measures are implemented, or
• the substance is known to be a germ cell mutagen, meeting the criteria for classification in the hazard class germ cell mutagenicity (category 1A or 1B) and appropriate risk management measures are implemented, or
• the substance is of low toxicological activity (a comprehensive and informative dataset showing no toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.
If a substance is known to have an adverse effect on sexual function and fertility, meeting the criteria for classification in the hazard class reproductive toxicity (category 1A or 1B: May damage fertility (H360F)), and the available data are adequate to support a robust risk assessment, then no further testing for sexual function and fertility shall be necessary.
If a substance is known to cause developmental toxicity, meeting the criteria for classification in the hazard class reproductive toxicity (category 1A or 1B: May damage the unborn child (H360D)), and the available data are adequate to support a robust risk assessment, then no further testing for developmental toxicity shall be necessary.’
None of the above-mentioned conditions is met. Therefore, the specific Column 2 adaption options are not adequate to generate the necessary information for the registered substance.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
The testing proposal is based on the results of a screening study for reproductive / developmental toxicity according to OECD guideline 422 and the lack of relevant effects in reproductive tissues and organs indicative of reproductive toxicity observed in subchronic (90-day) oral repeated dose toxicity (OECD 408) and prenatal developmental toxicity studies in rats and rabbits (OECD 414).
The following study is proposed: Extended one-generation reproductive toxicity study according to Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 (B.56 of the Commission Regulation on test methods or OECD guideline 443) in rats, oral route with the registered substance, specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation
- Dose level setting shall aim to induce some toxicity at the highest dose level
- Cohort 1A (Reproductive toxicity)
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation
For a justification of the test design, please refer to ‘Justification of study design’ below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals:
Ten weeks premating exposure duration for the parental (P0) generation as recommended by ECHA. This is the default value as specified by ECHA in recent decisions on OECD 443 studies.
- Basis for dose level selection:
The dose level setting shall aim to induce some toxicity at the highest dose level but not causing suffering or death. A recently performed combined repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD 422), a subchronic (90-day) oral repeated dose toxicity (OECD 408) and a prenatal developmental toxicity (OECD 414) studies in rats with the registered substance are available. Dose levels applied in the studies were 100, 300 and 1000 mg/kg bw/day. No systemic toxicity was observed in any of the studies. Consequently, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in all studies was set at the high-dose level of 1000 mg/kg bw/day.
- Inclusion/exclusion of extension of Cohort 1B:
This cohort is a standard requirement of an OECD guideline 443 study. Cohort 1B (Reproductive toxicity) will be included without extension to mate the Cohort 1B animals to produce the F2 generation.
Based on the available combined repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD 422), subchronic (90-day) oral repeated dose toxicity study (OECD 408) and prenatal developmental toxicity studies (OECD 414) in rats and rabbits, all conducted with the registered substance, no relevant effects or adverse changes in reproductive tissues and organs indicative for reproductive / developmental toxicity have been observed. Therefore, the extension to mate the Cohort 1B animals is not justified.
- Termination time for F2:
Not relevant as the production of the F2 generation is not planned.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B:
The Cohorts 2A and 2B are not planned to be included in the study design. There are no adverse effects in relation to reproductive / developmental toxicity observed in any of the studies mentioned above which might trigger the inclusion of Cohort 2A and 2B.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3:
The Cohort 3 is not planned to be included in the study design. There are no adverse effects in relation to reproductive / developmental toxicity observed in any of the studies mentioned above which might trigger the inclusion of Cohort 3.
- Route of administration:
The standard route of administration is the oral route.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals:
The standard species is the rat. It is intended to use the same rat strain as used in previous studies (Crl: WI(Han)) depending on the historical control database in the selected test facility. All other parameters will be selected as recommended in OECD guideline 443.

Species:

rat

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The selected study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.7, of the REACH Regulation (EC) No. 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Data on toxicity to reproduction (fertility) are available for alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Study with alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5)

The reproductive toxicity of alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) was tested in Wistar Han rats in a combined repeated dose toxicity study with the reproductive / developmental toxicity screening test according to OECD guideline 422 under GLP conditions (Shell, 2021). Groups of 10 animals per sex received doses of 100, 300 and 1000 mg/kg bw/day by daily oral gavage, 7 days a week for a minimum of 28 days. A similarly constituted control group was dosed with the vehicle (corn oil) only. Males were treated for 28-29 days whereas females that delivered were treated for 50-63 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females which failed to deliver or had a total litter loss were treated for 42-54 days. The following parameters and endpoints relevant for reproductive toxicity were evaluated: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care. In addition, a number of developmental parameters as well as endpoints indicative of repeated dose toxicity were investigated. The details of the assessment of developmental toxicity are summarised below under 'Additional information' in the section for effects on developmental toxicity and the repeated dose toxicity details are provided in IUCLID section 7.5.1.

All females had regular cycles of 4 to 5 days. Extend di-estrous occurred during the mating period in three females of the control group and two females of the mid-dose group (300 mg/kg bw/day) with a regular cycle during premating. One female at 300 mg/kg bw/day had an inconclusive cycle determination during the premating phase. Given their absence of a dose-related incidence, this finding did not indicate a relation with treatment. Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day. Mating index was not affected by treatment. The mating indices were 90, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One female of the control group did not mate. All paired females showed evidence of mating within 4 days, except one female at 300 mg/kg bw/day for which mating took 13 days. Hence, precoital time was not affected by treatment with the test item. Number of implantation sites was considered not to be affected by treatment. The mean number of implantation sites were 11.0, 8.9, 12.9 and 12.1 for the control, 100, 300 and 1000 mg/kg bw/day. The relatively low mean number of implantation sites at 100 mg/kg bw/day was attributed to the low number of implantation sites in three females (4, 1 and 2 implantation sites, respectively). In the absence of a dose-related incidence, the relatively low mean number of implantation sites at 100 mg/kg bw/day was considered not to be related to treatment with the test item. One female at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment with the test item. The fertility indices were 100, 90, 100 and 90% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. It was considered not to be affected by treatment of the animals. Gestation index and duration of gestation were not affected by treatment with the test item up to 1000 mg/kg bw/day. The gestation indices were 100% for all groups. All pregnant females had 21-22 days gestation, except for one female at 100 mg/kg bw/day which only had 19 days of gestation (her litter consisted of 1 pup only). Given the incidental occurrence and lack of a dose-related trend, no toxicological relevance was attributed to this early delivery. No signs of difficult or prolonged parturition and no deficiencies in maternal care were noted among the pregnant females.

Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) of = 1000 mg/kg bw/day for reproductive toxicity was determined.

Studies in the AE category

Studies investigating toxicity to reproduction are available for the following AE substances (Table 1):

Table 1

CAS No.	EC No.	Substance	Screening study (OECD 422)
			NOAEL reproduction/ fertility [mg/kg bw/day]	NOAEL systemic [mg/kg bw/day]
Linear subgroup
26183-52-8	500-046-6	Decan-1-ol, ethoxylated	= 950	= 950
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	= 1000	= 1000
9004-95-9	939-518-5	Hexadecan-1-ol, ethoxylated	= 1000	= 1000 (local: 300)
68439-49-6	939-518-5	Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO	= 1000	= 1000
9004-98-2	500-016-2	(Z)-9-Octadecen-1-ol ethoxylated	= 1000	= 1000
Mixed branched & linear subgroup
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	300	300
160901-19-9	500-457-0	Alcohols, C12-13, branched and linear, ethoxylated	= 1000	= 1000
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	= 1000	= 1000

Conclusion on toxicity to reproduction (fertility)

The data on fertility available for alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) is consistent with the overall toxicity to reproduction data for AE substances. The following NOAELs were set:

Oral (rat, m/f, OECD 422): NOAEL (reproduction) = 1000 mg/kg bw/day

Oral (rat, m/f, OECD 422): NOAEL (systemic toxicity) = 1000 mg/kg bw/day

Oral (rat, m/f, OECD 422): NOAEL (local toxicity) = 1000 mg/kg bw/day

 

For a detailed evaluation of the toxicity to reproduction potential of the substances in the AE category, please refer to the category justification attached to the category object.

 

Effects on developmental toxicity

Description of key information

Oral (rat, OECD 414): NOAEL (developmental toxicity) = 1000 mg/kg bw/day

Oral (rat, OECD 414): NOAEL (teratogenicity) = 1000 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (developmental toxicity) = 400 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (teratogenicity) = 400 mg/kg bw/day

 

Conclusion based on data obtained with alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) and considering all the available data on developmental toxicity in the Alcohol Ethoxylates (AE) category, in a Weight-of-Evidence approach.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Sep 2021 - 10 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

no

Principles of method if other than guideline:

The humidity values were outside the targeted range for 5 days with a maximum of 80% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study, the deviation was considered acceptable.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wl(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 - 15 weeks
- Weight at study initiation: 177 - 254 g
- Fasting period before study: not applicable
- Housing: Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany)
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 - 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 41 - 80%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Sep 2021 To: 1 Oct 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of the test material was mixed with the vehicle. The dose volume for each animal was based on the most recent body weight measurement and the dose formulations were stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 25 mg/mL (for 100 mg/kg bw/day), 75 mg/mL (for 300 mg/kg bw/day), 250 mg/mL (for 1000 mg/kg bw/day)
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analyses confirmed that formulations of the test item in corn oil were prepared accurately and homogenously.
In the Group 1 formulations prepared for use in Weeks 1 and 2, no test item was detected.
The concentrations analyzed in the Groups 2, 3, and 4 formulations prepared for use in Week 1 and Week 2 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115% of target concentration).
The formulations of Groups 2 and 4 prepared for use in Weeks 1 and 2 were homogeneous (i.e. coefficient of variation = 10%).

Details on mating procedure:

- Impregnation procedure: not reported
Females were time-mated and arrived at the testing facility as such. Day 0 of gestation is the day of mating, and was decribed as Day 0 post-coitum.

Duration of treatment / exposure:

Day 6 - 20 post-coitum

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy on Day 21 post-coitum

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels in this study were selected to be 100, 300, 1000 mg/kg/day, based on a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening with oral exposure of Alcohols, C12-15, Ethoxylated <= 2.5 EO in rats, (Test Facility Study No. 20219689).
- Fasting period before blood sampling for (rat) dam thyroid hormones: No
- Time of day for (rat) dam blood sampling: Day 21 post-coitum

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy, performed directly post dosing.

CHECK FOR MORTALITY: Yes
- Time schedule: At least twice daily beginning on arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: on a regular basis throughout the study (monitored by visual inspection).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-coitum
All animals from all groups were subjected to a gross necropsy. All gross lesions were collected.
- Organs weighed: thyroid
- Tissues collected for histopathology: thyroid gland and macroscopic abnormalities.
- Fixative: 10% buffered formalin
- Embedding media: paraffin
- Thickness of sections: 2 - 4 µm
- Staining: hematoxylin and eosin

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: 1.0 mL
- Parameters checked: triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH).

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes (all per litter)
- Body weight: Yes, all per litter
- Sex: Yes, all per litter

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. All pairwise comparisons were conducted against the control group (Group 1).
The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and Dunn’s test, ANCOVA and Fisher's exact test.

Indices:

Pregnancy rate (%): (No. of pregnant females)/(No. of mated females) * 100
Male fetuses (%): (No. of male fetuses)/(No. of fetuses) * 100
Female fetuses (%): (No. of female fetuses)/(No. of fetuses) * 100
Pre-implantation loss (%): (No. of corpora lutea – No. of implantations)/(No. of corpora lutea) * 100
Post-implantation loss (%): (No. of implantations – No. of live fetuses)/(No. of implantations) * 100
Litter % of fetuses with abnormalities: (No. of fetuses in litter with a given finding)/(No. of fetuses in litter examined) * 100

Historical control data:

Historical control data regarding fetal pathology was provided and can be found in Attachment 3 under "Overall remarks, attachments".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of toxicity that were noted were salivation in 1/22, 12/22 and 22/22 animals of the 100, 300 and 1000 mg/kg bw/day group. This was considered treatment-related, but non-adverse as they were a physiological response rather than a sign of systemic toxicity.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant difference was observed regarding body weight between the control and 100 mg/kg bw/day group.
At 300 mg/kg bw/day, slightly lower body weight gain compared to control was observed on Day 12 - 15 post-coitum. After this period and over the whole dosing period, comparable body weight gain was noted. Furthermore, body weight gain corrected for gravid uterus weight was comparable to control.
In the 1000 mg/kg bw/day group, slightly lower mean body weight gain was noted at start of dosing
(Days 6 - 9 post-coitum) and end of dosing (Days 18 -21 post-coitum), with mean body weight remaining slightly lower than for controls from Day 9 post-coitum onwards (not statistically significant). Terminal body weight was 4% lower compared to control (not statistically significant). Mean body weight gain corrected for mean gravid uterus weight was 7% compared to 13% in control.

As the differences were slight and below 10%, they were not considered adverse.

Summarized results can be found in Attachment 2 (tables) and 1 (figures) under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment-related difference was observed regarding food consumption between the control and 100 mg/kg bw/day group.
At 300 mg/kg bw/day, mean food consumption was lower compared to control from Day 12 post-coitum onwards (reaching statistical significance over Days 18-21 post-coitum with a maximum of -9% in the Day 18 - 21 interval).
In the 1000 mg/kg bw/day group, mean food consumption was lower compared to control throughout the dosing period. The difference was statistically significant from Day 6 - 15, with a maximum difference of -15% compared to the control in the first interval (Days 6 - 9 post-coitum). Overall food intake during the dosing period was 10% lower in high dose dams, compared to control.

As the differences were slight, they were not considered adverse.

Summarized results can be found in Attachment 2 (tables) and 1 (figures) under "Overall remarks, attachments".

Food efficiency:

not examined

Description (incidence and severity):

not applicable

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No differences were observed regarding water consumption between the control and treatment groups up to and including the highest dose level.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related differences were observed regarding T3, T4 and TSH between the control and 100 mg/kg bw/day group.
At 300 and 1000 mg/kg bw/day, decreased serum levels of total T3 were noted (0.85x and 0.75x of control, respectively, corresponding to 0.381 ng/mL for the 300 mg/kg bw/day group and 0.336 ng/mL for the 1000 mg/kg bw/day group compared to 0.45 ng/mL in the control group). Although, both means remained within the available historical control data (total T3 (ng/mL): mean = 0.439; P5 – P95 = 0.280 – 0.616 (n = 263) in the period of 2020 - 2021), a dose response is present. Therefore, a decrease in total T3 level was considered test item related. However, as no other associated effect was noted and as both mean values were within the historical data range, the effect on total T3 level in serum is considered as non-adverse.


Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related differences were observed regarding thyroid weights between the control and treatment groups up to and including the highest dose level.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related differences were observed during gross pathology between the control and treatment groups up to and including the highest dose level.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related differences were observed in histopathology examination of the thyroid between the control and treatment groups up to and including the highest dose level.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions occurred in this study.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant difference regarding pre- and post-implantation loss was observed between the control and treatment groups up to and including the highest dose level.
At 1000 mg/kg bw/day, mean pre-implantation loss was higher compared to the control group (12.71% vs 7.65% in the control group; not statistically significant). However, as the treatment started on Day 6 post-coitum (after implantation), the effect is not considered treatment-related.
Post-implantation loss was higher in all treatment groups (7.54, 6.51 and 6.15% for the 100, 300 and 1000 mg/kg bw/day group, respectively), compared with 3.90% in control; none of these differences was statistically significant. However, all mean values remained within the historical data (mean: 5.2, P5 – P95 = 2.1 – 9.0 (n = 706)) and no dose-response was observed. Therefore, the higher post-implantation loss is considered not related to treatment with the test-item.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding total litter losses by resorption was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Early or late resorptions:

no effects observed

Description (incidence and severity):

No treatment-related differences regarding resorptions were observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Dead fetuses:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding the number of dead fetuses was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding the pregnancy duration was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding the number of pregnant females was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects were observed up to and including the highest dose level.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related differences regarding fetal body weights were observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding the number of live offspring was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding sex ratio was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding litter size was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No treatment-related difference regarding anogenital distance was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related external malformations and variations were observed.
The malformations that occurred in this study were a generalized subcutaneous edema in one fetus of the 100 mg/kg bw/day group and a cleft palate in one fetus of the control group. These incidences were considered to be single occurrences and therefore not treatment-related.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related skeletal malformations were observed.
Skeletal malformations that were not considered treatment-related were bent humeri (in 1 control fetus, 1 fetus of the 300 mg/kg bw/day group), multiple malformations relating the lower jaw, skull, vertebrae and sternum (1 additional fetus of the 300 mg/kg bw/day group). The observed sternal anomaly above (sternoschisis) also occurred in the fetus with generalized subcutaneous edema at 100 mg/kg bw/day. Due to single occurrence and lack of a dose-response, a relationship with the test-item was considered unlikely.
Skeletal variations that were observed in all treatment groups were increased incidences of misaligned ilia (pelvic girdle), but the difference to the control group did not reach statistical significance and occurred without any dose-response. Therefore, it was not considered related to treatment.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related visceral malformations were observed in this study.
Only one visceral malformation was observed in 1 control female (dilated brain ventricles), this was considered spontaneous in origin.
The variations that were noted affected the liver (supernumerary lobes) and ureters (convoluted and dilatation) at low incidences or in isolated cases, which did not indicate that they were treatment-related.

Summarized data can be found in Attachment 2 under "Overall remarks, attachments".

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to and including the highest dose level.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The present study was conducted under GLP and according to OECD test guideline 414 (2018).
Under the conditions of the study, administration of the test substance once daily by oral gavage for Days 6 - 20 post coitum was well tolerated in pregnant rats at levels up to 1000 mg/kg bw/day. No adverse effects on dams were observed up to and including the highest dose level. No test item-related adverse findings were observed causing developmental toxicity. Therefore, the NOAEL for maternal systemic and developmental toxicity was set at 1000 mg/kg bw/day. No teratogenicity was observed (NOAEL = 1000 mg/kg bw/day).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Oct 2021 - 10 Jan 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose range finder study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

limited number of litters evaluated in the control group (15 instead of 16) due to mortality and non-pregnant animals (see results fields for details).

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: 2835 – 4410 g
- Fasting period before study: no
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet: KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Switzerland (25 g at arrival, on subsequent days 140 - 160 g). In addition, pressed hay (daily allotment; Tecnilab-BMI B.V., Someren, The Netherlands) and fresh fruits and/or vegetables (at least twice weekly) were provided during the study period.
- Water: tap water, ad libitum
- Acclimation period: 5 - 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 19
- Humidity (%): 47 - 61
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Oct To: 26 Nov 2021

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of the test material was mixed with the vehicle. The dose volume for each animal was based on the most recent body weight measurement and the dose formulations were stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 66.67 mg/mL (100 mg/kg bw/day), 166.67 mg/mL (250 mg/kg bw/day), 266.67 mg/mL (400 mg/kg bw/day)
- Amount of vehicle (if gavage): 1.5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity by ultra performance liquid chromatography with mass spectroscopy (UPLC-MS).
The mean concentration analyzed in the Group 2 formulation prepared for use in Week 1 was in agreement with the target concentration (i.e. within or equal to 90-110% of target concentration). For the formulations of Group 3 and Group 4, the mean concentrations were each 88% of target. It was considered that these formulations were prepared correctly, since the QC samples measured in the same series showed comparable accuracies (i.e., mean recoveries of 90% and 85% of target concentration for the 67 mg/mL and the 267 mg/mL QC sample, respectively).

In one sample of formulations from Group 1 (vehicle), a small response at the retention time of the ammonium adduct of C13E5 was observed for the Week 1 formulation. In the chromatogram of the other sample from the Group 1 formulation prepared for use in Week 1 and in the chromatograms of both samples from the Group 1 formulation prepared for use in Week 4, no test item was detected. Therefore, the small response in this one sample was not considered to derive from the formulation. It was considered the small response in the Week 1 sample of Group 1 was not derived from the formulation, since no response was observed in the duplicate Week 1 sample.

The formulations of Groups 2 and Group 4 prepared for use in Week 1 and Week 4 were homogeneous (i.e., coefficient of variation = 10%).

In Week 4, the pH of the dosing formulations was assessed, the pH was 8.31 - 8.42 for all dose groups.

Details on mating procedure:

- Impregnation procedure: not reported
Females were time-mated and arrived at the testing facility as such.

Duration of treatment / exposure:

Day 7 - 28 post-coitum

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy at Day 29 post-coitum

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

400 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on a dose range finding experiment (Test Facility Study No. 20288391). In this Dose Range Finder, dose levels of 250, 500 and 750 mg/kg bw/day were tested in pregnant rabbits. Test item-related mortality was observed in 1/6 and 3/6 animals at respectively 500 and 750 mg/kg bw/day. These animals were sacrificed in extremis for animal welfare reasons based on prolonged absent food in combination with severe body weight loss and/or based on severe clinical signs, consisting of some type of episode (i.e. lying on side, decreased activity, extensor rigidity and absent withdrawal reflex of one or both fore and/or hindlimb(s), partly closed eyes, ataxia and/or (moderate to severely) uncoordinated movements). At 250 mg/kg bw/day, reduced food consumption was noted (approximately 12% lower overall).

Maternal examinations:

CHECK FOR MORTALITY: Yes
- Time schedule: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily; starting on Day 7 post-coitum up to the day prior to necropsy. 0 to 1 hours postdose and 2 to 4 hours postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum, calculated for the intervals Days 7-9, body weight gain was calculated for the intervals Day 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29, and 7-29 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: on regular basis throughout the study (monitored by visual inspection)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. The digestive tract (esophagus, stomach, large intestine (cecum, colon, rectum, appendix), small intestine (ileum, jejunum, duodenum), sacculus rotundus were collected and fixed in the appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yesa
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Blood sampling:

No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter (heads of ~50% of fetuses were fixed in Bouin’s solution for soft-tissue examination, tissues without variations or malformations were discarded. Tissues with variations or malformations were stored. Heads of other ~50% of fetuses were examined by mid-coronal slice)

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. All pairwise comparisons were conducted against the control group (Group 1).
The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and
Dunn’s test, ANCOVA, Fisher's exact test.

Indices:

Pregnancy rate (%): (No. of pregnant females)/(No. of mated females) * 100
Male fetuses (%): (No. of male fetuses)/(No. of fetuses) * 100
Female fetuses (%): (No. of female fetuses)/(No. of fetuses) * 100
Pre-implantation loss (%): (No. of corpora lutea – No. of implantations)/(No. of corpora lutea) * 100
Post-implantation loss (%): (No. of implantations – No. of live fetuses)/(No. of implantations) * 100
Litter % of fetuses with abnormalities: (No. of fetuses in litter with a given finding)/(No. of fetuses in litter examined) * 100

Historical control data:

Historical control data regarding maternal pregnancy data and fetal pathology was provided and can be found in Attachment 3 in the attached background material.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 100 mg/kg bw/day group, 1 female showed slight uncoordinated movements on Day 27 post-coitum, 2-4 h post-dose, and and 2/19 showed decreased activity on Day 19 and 27 post-coitum.
In the 250 mg/kg bw/day group, 1 female showed extensor rigidity of both hind limbs, lying on the side, prostrate, erected fur and/or hunched posture on individual days between Days 23 - 28 post-coitum, mostly 2-4 hours post-dose. This female was sacrificed in extremis on Day 24 post-coitum. Decreased activity was noted in 9/19 animals on individual days between Days 20-28 post-coitum, mostly 2-4 hours post-dose.
In the 400 mg/kg bw/day group, two females showed behavioral episodes on Days 20 and 22 post-coitum, respectively, 2-4 hours post-dose. This episode consisted of a combination of the following clinical signs: severely uncoordinated movements with extensor rigidity of both hind limbs, rolling and/or shallow breathing, decreased activity, limited usage (moderate) of both hind limbs, severe lameness of both hind limbs, abnormal gate, lying on side, difficulty raising and/or abnormal respiratory rate. Extensor rigidity of both hind limbs, lying on the side, prostrate, erected fur and/or hunched posture was also noted in these 2 females as well as 3 more females of this dose group on individual days between Days 23 - 28 post-coitum, mostly 2-4 hours post-dose. Decreased activity was noted in 12/19 animals on individual days between Days 20-28 post-coitum, mostly 2-4 hours post-dose.

Summarized results can be found in Attachment 2 in the attached background material.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

3 animals of the control group, 2 animals of the 100 mg/kg bw/day group, 3 animals of the 250 mg/kg bw/day group and 3 animals of the 400 mg/kg bw/day group died prematurely during the study.
7 of these 11 deaths were considered test-item related (1 at 100 mg/kg bw/day, 3 at 250 mg/kg bw/day, 3 at 400 mg/kg bw/day):
The animal of the 100 mg/kg bw/day group was sacrificed in extremis for animal welfare reasons on Day 17 post-coitum. The animal had limited to absent food consumption for 7 consecutive days and slight body weight loss (4% compared to the highest recorded body weight on site). The animal did not show any clinical signs and no abnormal findings were noted at necropsy. Ectopic spleen tissue and clear watery cyst on the oviduct were noted. Both these macroscopic findings are within the range of background findings to be expected for rabbits of this age and strain. Although a relationship to treatment with the test item could not be excluded, since mortality due to prolonged absent food consumption was also noted in one female of the control group and no effects on food consumption were noted in the remaining animals at 100 mg/kg bw/day, this death was not taken into account for determining the NOAEL. At early termination, this animal was pregnant with normal implantations for the duration of pregnancy.
The 3 animals of the 250 mg/kg bw/day group were sacrificed in extremis on Days 17, 24 and 20 post-coitum. 2 of the animals had limited to absent food consumption for 7 consecutive days and were noted with slight to moderate body weight loss (respectively 3 and 5% compared to the highest recorded body weight on site). The third animal had severe body weight loss (-11% compared to the highest recorded body weight). Its food consumption was lower than for the control group but stable with the exception of Days 12-16 post-coitum and 21-24 post-coitum. It was thin from Day 20 post-coitum and erected fur was noted on Days 23 and 24 post-coitum. Moreover, decreased activity and/or lying on side were noted for this animal on Days 21, 23 and 24 post-coitum, 0-1 and/or 2-4 hours post-dose. At necropsy, gelatinous watery content of the stomach was noted, as well as a small thymus. No abnormalities regarding pregnancies were noted in any of these animals.
For the 400 mg/kg bw/day group, the three animals that were sacrificed prematurely on Day 20, 17 and 19 post-coitum after limited to absent food consumption for =7 consecutive days. They had slight to moderate body weight loss (1 - 6% compared to the highest recorded body weight on site). At necropsy, no abnormalities were noted for these animals and no abnormalities regarding pregnancies were noted in any of these animals.

2/11 deaths were related to gavage problems (1 control animal, 1 animal of the 100 mg/kg bw/day group). The control animal was found dead on Day 22 post-coitum. Red discharge from the nose and mouth was noted shortly prior to its death, as well as labored breathing and vocalizations. At necropsy, dark brown foci were noted on the caudal lobe of the right lung. In addition, clear watery cysts on both oviducts were noted, which is a finding that is within the range of background findings to be expected for rabbits of this age and strain. This animal was pregnant and had 10 dead fetuses in utero at necropsy. The animal of the 100 mg/kg bw/day group was sacrificed in extremis for animal welfare reasons on Day 22 post-coitum, as it was noted with labored breathing and pale skin immediately after dosing and blood was noted on the gavage tube. At necropsy, this animal was noted with dark red foci on - as well as a perforation of the caudal lobe of the right lung. In addition, dark red discoloration of the mammary gland was noted. This animal was pregnant and had 9 live fetuses in utero at necropsy.

The two additional deaths in the control group were 1 sacrifice in extremis on Day 11 post-coitum (the animal had not eaten for 8 consecutive days and lost 7% of its body weight compared to the highest recorded body weight on site) and 1 sacrifice in extremis on Day 19 post-coitum (the animal had limited to absent food consumption for 8 consecutive days and was noted with moderate body weight loss of 6%). No macroscopic abnormalities were noted at necropsy. Both animals were pregnant with normal implantations for the duration of pregnancy.

Summarized results can be found in Attachment 2 in the attached background material.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No difference was observed regarding body weight between the control and 100 mg/kg bw/day group. Body weight gain corrected for gravid uterus was slightly lower in this group compared to the control group without reaching statistical significance, but because the change was minor, it was not considered adverse.
At 250 mg/kg bw/day, slightly lower body weight gain was observed over Days 9-12 and 15-18 post-coitum (-49.7% and -80.1% compared to the control). None of the differences reached statistical significance. Overall body weight gain, body weight at the end of the dosing period and body weight gain corrected for gravid uterus weight were comparable to control. As body weight gain over the entire dosing period was comparable to the control group, the effect was not considered adverse.
In the 400 mg/kg bw/day group, body weight gain was lower throughout most of the dosing period, resulting in lower overall body weight gain (254 g vs. 391 g in control). However, the overall difference did not reach statistical significance. Mean body weight loss was observed over Days 9-12 post-coitum (-9.2 g), but this was not statistically significant. Body weight gain corrected for gravid uterus weight was slightly lower compared to control. Body weight at the end of the Dosing Period was comparable to control. The lower overall body weight gain was considered to be an adverse finding.

Summarized results can be found in Attachment 1 (Figures) and Attachment 2 (Tables) in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day, food consumption was slightly lower in intervals Days 9-12 and 21-27 post-coitum, which resulted in an overall food intake during the Dosing Period that was 7% lower than control. None of the differences were statistically significant. At 250 mg/kg bw/day, mean food consumption was lower from Day 9-24 post-coitum, but the difference did not reach statistical significance. Overall food intake during the dosing period was 6% lower compared to the control group (not statistically significant). Because of the minor changes in the 100 and 250 mg/kg bw/day group, the findings were not considered adverse.
At 400 mg/kg bw/day, mean food consumption was reduced (maximum of -32% compared to the control group) throughout the dosing period. The difference reached statistical significance over Days 9-21 post-coitum. Overall food intake during the dosing period was 18% lower that the control.

Summarized results can be found in Attachment 1 (Figures) and Attachment 2 (Tables) in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No differences were observed regarding water consumption between the control and treatment groups up to and including the highest dose level.

Summarized results can be found in Attachment 2 in the attached background material.

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant differences were observed regarding gross pathology between the control and treatment groups up to and including the highest dose level.
In 1-4 females of the control and treatment groups, dark foci on the stomach were noted. There was no dose-related trend observed and therefore these findings were considered not test item-related.

Summarized results can be found in Attachment 2 in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions occurred during the study.

Summarized results can be found in Attachment 2 in the attached background material.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant difference regarding pre- and post-implantation loss was observed between the control and treatment groups up to and including the highest dose level. Pre-implantation loss in Groups 1 (18.1%) and 3 (16.9%) were outside the historical control range (mean: 6.1%, P5 - P95: 1.6 - 12.7%), but as dosing with test item was initiated only after implantation, any effects on this parameter would not be considered test item related.

Summarized results can be found in Attachment 2 in the attached background material.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No total litter losses by resorption were observed in the control or treated groups. .

Summarized results can be found in Attachment 2 in the attached background material.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No difference regarding early or late resorption was observed between the control and treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 in the attached background material.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed for animals surviving until scheduled necropsy on Day 29 post-coitum.

Summarized results can be found in Attachment 2 in the attached background material.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No difference regarding the pregnancy duration was observed between the control and treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 in the attached background material.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

4/22 females of the control group, 2/22 females in the 100 mg/kg bw/day group, 3/22 females in the 250 mg/kg bw/day, 1/22 females in the 400 mg/kg bw/day group were not gravid at time of scheduled necropsy. Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 15, 18, 16 and 18 females with viable litters in the control, 100, 250 and 400 mg/kg bw/day groups, respectively.

Summarized results can be found in Attachment 2 in the attached background material.

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects were observed at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Remarks on result:

other: At the high dose level, lower overall body weight gain and food consumption were noted and considered adverse findings.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No differences regarding fetal body weights were observed between the control and treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 in the attached background material.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No difference regarding the number of live offspring was observed between the control and treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 in the attached background material.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No toxicologically relevant difference regarding sex ratio was observed between the control and treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 in the attached background material.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant differences were observed regarding litter sizes between control and treatment groups up to and including the highest dose group.
At 400 mg/kg bw/day, the number of live fetuses was lower (7.7 vs 8.6 in control). The difference did not reach statistical significance and was considered the consequence of the lower number of corpora lutea and hence the lower number of implantation sites at this dose, and was therefore considered not to be test item-related.

Summarized results can be found in Attachment 2 in the attached background material.

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related external malformations and variations were observed.
The malformations that occurred in this study were malrotated hindlimbs in one fetus of the 100 mg/kg bw/day group, multiple malformations affecting the entire body in one fetus of the 250 mg/kg bw/day group and a cleft palate, hyperflexed forepaws and malrotated hindpaws in one fetus of the 400 mg/kg bw/day group. These incidences were considered to be single occurrences and therefore not treatment related.

Summarized results can be found in Attachment 2 in the attached background material.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were two (1), four (3), one (1) and three (3) fetuses (litters) with skeletal malformations in the control, 100, 250 and 400 mg/kg bw/day groups, respectively. The fetus of the 250 mg/kg bw/day group that had external malformations had associated multiple skeletal malformations and the fetus of the 400 mg/kg bw/day group had a cleft palate and palatine split.
All other malformations, concerning thoracic, lumbar or caudal vertebrae, sternebrae or nasal bones occurred only once or twice in a group. The low incidences and group distribution of these diverse malformations do not indicate a test item-relationship and therefore they were considered chance findings.

All skeletal variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any effects from treatment with the test item. All skeletal variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any effects from treatment with the test item.

Summarized results can be found in Attachment 2 in the attached background material.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were two (2), two (2), three (2) and three (3) fetuses (litters) observed in the control, 100, 250 and 400 mg/kg bw/day groups, respectively, with visceral malformations affecting the subclavian artery, kidneys, heart and major blood vessels, lung, diaphragm, brain and eye.
The most common was retro esophageal right subclavian artery (1 fetus of the control group, 1 fetus of the 100 mg/kg bw/day group, 1 fetus of the 400 mg/kg bw/day group).
As the incidences were evenly distributed among the groups, the finding not considered treatment related.
All other malformations occurred only once or twice in a group of which in the high-dose group several malformations (affecting brain, lung and diaphragm) concerned the already externally malformed fetus. The low incidences and group distribution of these diverse malformations do not indicate a test item-relationship and therefore they were considered chance findings.

Visceral variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any effects from treatment with the test item.

Summarized results can be found in Attachment 2 in the attached background material.

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to and including the highest dose level.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The present study was conducted under GLP and according to OECD test guideline 414 (2018). Time-mated female New Zealand White rabbits were treated with the test substance once daily by oral gavage for Days 7-28 post-coitum.Mortality and adverse clinical signs were observed at 250 and 400 mg/kg bw/day and lower overall body weight gain and food consumption at 400 mg/kg bw/day. Therefore, the NOAEL for the test substance regarding maternal systemic toxicity was set at 100 mg/kg bw/day. No effects were noted regarding developmental toxicity. Therefore, the developmental NOAEL was set at 400 mg/kg bw/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 Sep - 14 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

Main OECD 414 study

Qualifier:

no guideline required

Principles of method if other than guideline:

No guideline was applicable since the study was intended for dose selection purposes only. Therefore, various examinations were not performed (e.g. soft tissue and skeletal malformations, head examination and anogenital distances of fetuses.
The humidity values that were outside the targeted range occurred for 6 days with a maximum of 79% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. 2 animals of the 750 mg/kg bw/day and 1 animal of the 500 mg/kg bw/day group were not dosed for one day each (Day 9, 16 and 19, respectively) based on clinical signs.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Age at study initiation: 17 - 19 weeks
- Weight at study initiation: 3048 – 4516 g
- Fasting period before study: not applicable
- Housing: individually housed in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm)
- Diet: pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Switzerland). Animals received 25 g pelleted diet on the day of arrival and 140-160 g pellets on subsequent days. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and fresh fruits and/or vegetables were provided during the study period.
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean of 19 °C
- Humidity (%): 49 - 79
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: From: 16 Sep 2021 To: 14 Oct 2021

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared at least weekly. The appropriate amount of the test material was mixed with the vehicle. The dose volume for each animal was based on the most recent body weight measurement and the dose formulations were stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 166.67 mg/mL (250 mg/kg bw/day), 333.33 mg/mL (500 mg/kg bw/day), 500 mg/mL (750 mg/kg bw/day)
- Amount of vehicle: 1.5 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the formulations under test conditions were already demonstrated in a previous study from the laboratory (analytical method development and validation study, Test Facility Study No. 20309107).

Details on mating procedure:

The animals were mated by the supplier on the 15th of September 2021 and arrived in the testing facility one day after. The day of mating is considered Day 0 post-coitum (Day 0 of gestation).

Duration of treatment / exposure:

Day 7 - 28 post-coitum

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy at Day 29 post-coitum

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

750 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

6 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a tolerability study with the test item in rabbits by oral gavage (Test Facility Study No. 20288390). At 1000 mg/kg bw/day, one female was euthanized for humane reasons as it was observed with an increased respiratory rate and uncoordinated movement after dosing, body weight loss up to 8% and reduced food consumption. The remaining two animals at this dose level showed weakness or hunched posture, body weight loss between 5 - 9% and reduced food consumption. Therefore, this dose level was considered to exceed the Maximum Tolerated Dose level. At 500 mg/kg bw/day, no clinical signs were observed. For two of the females, body weight loss was observed (occasional body weight loss or continuous loss up to 5%) as well as reduced food consumption during the first days of treatment.

Maternal examinations:

CHECK FOR MORTALITY: Yes
- Time schedule: twice daily throughout the study.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily post-dose, starting on Day 7 post-coitum onwards up to and including the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum, calculated over each sequent interval and over the total dosing period. Corrected body weight gain will be determined based on Day 7 and Day 29 post-coitum body weights and the gravid uterus weight.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum by intravenous injection of sodium pentobarbital.
- Examinations: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) will be subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Blood sampling:

No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: No
- Body weight: Yes, all per litter

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. All pairwise comparisons were conducted against the control group (Group 1).
The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and Dunn’s test and Fisher's exact test.

Historical control data:

Not provided

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically significant clinical signs were noted in animals of the control and 250 mg/kg bw/day group. In the 500 mg/kg bw/day group, erected fur was noted incidentally in 4/6 animals on individual days between Day 14 and 28 post coitum. In addition, abnormal breathing sounds and/or labored breathing were noted incidentally in 2/6 animals between Day 8 and 28 post-coitum. As no animals of the 750 mg/kg bw/day group survived until scheduled sacrifice, the clinical signs are discussed under "Mortality".

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

No animals of the control and 250 mg/kg bw/day group died prematurely during the study.
One animal of the 500 mg/kg bw/day group was sacrificed in extremis for animal welfare reasons on Day 19 post-coitum as it had limited to absent food consumption for 7 consecutive days and
it had lost 9% of its weight compared to the highest body weight measured
In the 750 mg/kg bw/day group, all animals were sacrificed before scheduled necropsy. 3/6 animals were sacrificed in extremis for animal welfare reasons (one animal day 9 and 2 animals on Day 16 post coitum). All animals showed some type of episode (decreased activity, increased respiratory rate and extensor rigidity of fore- and hindlimbs, partly closed eyes, ataxia and moderately uncoordinated). Moreover, all three animals showed decreased food consumption and body weight loss and were therefore sacrificed.
1/6 animals of the 750 mg/kg bw/day group was sacrificed in extremis for animal welfare reasons on Day 9 post-coitum. During the oral gavage procedure, red fluid on the dosing tube was noted. Upon further investigation, labored breathing, abnormal breathing sounds and shallow breathing as well as pallor skin was noted and the animal was sacrificed. At necropsy, red watery fluid in the thoracic cavity was noted, as well as dark red discoloration of the right lung. This death was considered related to the gavage procedure and not to treatment with the test item.
The 2 remaining animals of this group were sacrificed as well on Day 16 post-coitum, in consultation with the Sponsor. For one animal, erected fur was noted between Day 14 and 16 post-coitum, as well as decreased activity on Day 16 post-coitum. Food consumption was reduced for these animals from the start of the treatment onwards. Slight body weight loss was noted for these animals between Days 7 - 9 and 9 - 12 post-coitum, which appeared to stabilize to slight body weight gain between Days 12 -15 post-coitum. At necropsy, no toxicologically relevant macroscopic findings were noted for these animals.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight (gain) and body weight corrected for gravid uterus weight were within normal range in the control and 250 mg/kg bw/day group. In the 500 mg/kg bw/day group, body weight loss or lower body weight gain was observed between Day 7 - 18 post-coitum (reaching statistical significance between Days 9 -15 post-coitum and up to 5.5% body weight loss in individual animals). Body weight gain comparable to control was observed after this period. Overall body weight gain between Day 7 - 29 was statistically significantly lower than in concurrent controls. Although not statistically significant, body weight gain corrected for gravid uterus weight was lower compared to controls and showed a marked body weight loss (-294.6 g vs. - 89 g in concurrent controls).

Body weight changes and body weight gain differences of animals that died prematurely are summarized under "Mortalities".

Summarized results can be found in Attachment 1 (Figures) and Attachment 2 (Tables) under "Overall remarks, attachments" under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From the start of the treatment onwards, a dose-dependent reduction in food consumption was observed, starting at 250 mg/kg bw/day and up to absent food consumption for several days in individual animals at 500 mg/kg bw/day. Over the entire treatment period (Day 7-29), food consumption was circa 12% and 36% lower compared to concurrent controls at respectively 250 and 500 mg/kg bw/day.

Food consumption differences of animals that died prematurely are summarized under "Mortalities".

Summarized results can be found in Attachment 1 (Figures) and Attachment 2 (Tables) under "Overall remarks, attachments".

Food efficiency:

not examined

Description (incidence and severity):

not applicable

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Prominent lobular architecture of the liver was noted in 1/6 animals of the control group and multiple dark red foci on the lung were noted for 1/6 animals of the 500 mg/kg bw/day group. Clear watery cysts on the oviduct and/or ectopic splenic tissue were found within all treatment groups and the control and were within the range of background findings to be expected for rabbits of this age and strain. Any other remaining findings at necropsy were considered not toxicologically relevant.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Number of abortions:

not specified

Description (incidence and severity):

not applicable

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect on pre-implantation loss compared to the control group was observed in any treatment group. Post-implantation loss was higher compared to the control group in the 250 mg/kg bw/day group (10.32% vs. 6.61% in concurrent controls) and in the 500 mg/kg bw/day group (13.44% vs. 6.61% in concurrent controls). However, according to the main study (Study No. 20288392), the historical control data for post-implantation Loss (%/Litter) was: mean: 6.3, P5 - P95: 3.0 - 13.6. Therefore, both the values for the 250 and 500 mg/kg bw/day group are within the range of the historical control data and are considered within normal biological variation.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No significant difference regarding total litter losses by resorption was observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Early or late resorptions:

no effects observed

Description (incidence and severity):

No significant difference regarding early or late resorption was observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Dead fetuses:

no effects observed

Description (incidence and severity):

No significant difference regarding the number of dead fetuses was observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No significant difference regarding the pregnancy duration was observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All animals of the control and 500 mg/kg bw/day group were pregnant with viable fetuses. 1/6 animals of the 250 mg/kg bw/day group was not pregnant, but this was considered incidental.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: No difference was observed between the control and treatment group.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant differences regarding fetal body weights were observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No significant differences regarding viable fetuses were observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in sex ratio:

not examined

Description (incidence and severity):

not applicable

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Fetal body weights were similar across all groups.

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

no effects observed

Description (incidence and severity):

External examination of the fetuses did not show any abnormalities.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Skeletal malformations:

not examined

Description (incidence and severity):

not applicable

Visceral malformations:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including this dose level.

Remarks on result:

other: The higher dose level of 750 mg/kg bw/day could not be evaluated due to excessive maternal toxicity.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The present study was not conducted under GLP or according to any guideline as it was a dose range finding study. However, it was conducted similarly to OECD test guideline 414 (2018). In the present study, in time-mated female New Zealand White rabbits were treated with the test substance once daily by oral gavage for Days 7-28 post-coitum. Severe clinical signs were observed in the high dose group (750 mg/kg bw/day), wherefore all animals of this group were sacrificed prematurely. Decreased food consumption and body weight (gain) were noted in the 500 mg/kg bw/day group. In the 250 and 500 mg/kg bw/day group, increased postimplantation loss compared to the control groups were noted, but both values were within reference values for this strain. Therefore, the NOAEL for systemic toxicity was set at 250 mg/kg bw/day. No effects were noted regarding developmental toxicity. Therefore, the developmental NOAEL was set at 500 mg/kg bw/day as the high dose group could not be evaluated due to the severe maternal toxicity.

Based on the results of this Dose Range Finder and in consultation with the Sponsor, selected dose levels for the Main study (Test Facility Study No. 20288392) are 100, 250 and 400 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies performed with the registered substance. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.7, of the REACH Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Data on developmental toxicity are available for alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) as well as several member substances of the Alcohol Ethoxylates (AE) category.

Studies with alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5)

As briefly described under 'Additional information' in the section 'Effects on fertility' above, the reproductive and developmental toxicity of alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) was tested in Wistar Han rats in a combined repeated dose toxicity study with the reproductive / developmental toxicity screening test according to OECD guideline 422 under GLP conditions (Shell, 2021). The experimental details of the study are summarised above and in IUCLID section 7.5.1. Only information relevant for developmental toxicity is provided here. The following parameters relevant for developmental toxicity were evaluated: sex ratio and early postnatal pup development, i.e. mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormone T4 (post-natal day (PND) 14-16).

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item up to 1000 mg/kg bw/day. Post-implantation survival index was 90, 96, 91 and 94% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Live litter sizes were 9.7, 8.6, 11.7 and 11.3 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively and, hence, litter size was considered not affected. One litter in the control group, two litters at 100 mg/kg/day and one litter at 300 mg/kg/day consisted of 1 or 2 pups at first litter check. This was related to the lower number of implantation sites. However, in absence of a dose-related effect, these findings were considered not to be toxicologically relevant. The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. The live birth indices were 97, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Three pups in one litter of the control group were found dead at first litter check. No toxicological relevance was attributed to these dead pups since these mortalities occurred only in the control group. Viability indices were 99, 100, 98 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One pup of the control group and two pups at 300 mg/kg bw/day were missing on PND 2 or 3, which were most likely cannibalised. The loss of the only pup of one of the females lead to a total litter loss. In absence of a dose-related trend and at the incidence observed, no toxicological relevance was attributed to the missing pups or total litter loss. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected. The lactation indices were 98, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One pup of the control group was sacrificed in extremis on PND 7, which resulted in a total litter loss. No toxicological relevance was attributed to this pup or total litter loss since this occurred in the control group. No clinical signs occurred among pups that were considered to be related to treatment. For the pups in the control group and the mid-dose group (300 mg/kg bw/day) that were found dead at PND 1, missing on PND 2 or 3, or euthanised in extremis on PND 7, absence of milk in the stomach were noted on PND 1, 2, 3 or 7. In addition, for the pups that were missing on PND 3 or euthanized in extremis on PND 7, a dehydrated appearance was noted on PND 1 and 7. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant. Body weights of pups were considered to be affected by treatment. At PND 7 and 13, mean body weights of male pups were statistically significantly decreased (0.88 and 0.86x of control, respectively) at 1000 mg/kg bw/day. In addition, mean body weights of female pups at 1000 mg/kg bw/day were decreased (0.90x of control; not statistically significant) at PND 13. At PND 13, male and female pup weights of 3/9 litters at 1000 mg/kg bw/day were below the historical control range. In addition, the body weights of two pup of the low-dose group (100 mg/kg bw/day) were significantly lower compared to the control mean at PND 7 and 13 indicating a growth retardation. In the absence of a dose-related trend of the same order of magnitude, these single occurrences in both litters were not considered toxicologically relevant. Sex ratio, anogenital distance (absolute and normalized for body weight in male and female pups) and areola/nipple retention were considered not to be affected by treatment with the test item. For none of the examined male pups nipples were observed at PND 13. Serum levels of total T4 in male and female PND 14-16 pups were affected by treatment with the test item at 100, 300 and 1000 mg/kg bw/day. At 100, 300 and 1000 mg/kg bw/day, mean levels of total T4 were increased to 1.28, 1.18 and 1.19x of control, respectively, in male PND 14-16 pups (statistically significant at 100 mg/kg bw/day only) and to 1.42, 1.26 and 1.33x of control, respectively, in female PND 14-16 pups (statistically significant in all treatment groups). The mean values remained within the historical control range. No macroscopic findings were noted among pups that were considered to be related to treatment. However, under the conditions of this screening study no adverse effect was observed that could be linked to the increase of total T4 and therefore this increase was not taken into account when determining the developmental NOAEL. No milk in the stomach was noted for two control pups only that died preterm. No indication of a teratogenic effect was observed at any dose level.

Based on reduced pup body weights, a No-Observed-Adverse-Effect-Level (NOAEL) of 300 mg/kg bw/day for developmental toxicity was determined.

 

Alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) was administered to female Wistar (Crl: WI(Han) rats in a Prenatal developmental toxicity study according to OECD guideline 414 under GLP conditions (Shell, 2022a). Groups of 22 dams were administered doses of 100, 300 and 1000 mg/kg bw/day by oral gavage, 7 days a week on gestation day 6 - 20. The control group was treated according to the same protocol and received the vehicle (corn oil) only. All the females were time-mated and the day of mating was gestation day 0 (Day 0 post-coitum). The dams were sacrificed on gestation day 21.

The following parameters were recorded in the dams: mortality/moribundity, clinical signs, body weight and food consumption, water consumption (monitored by visual inspection), thyroid weight, measurement of thyroid hormones (T3, T4 and TSH), macroscopic examination, gravid uterus weight, number of corpora lutea, number of implantations, number of early and late resorptions, and number of live and dead fetuses. The following parameters were recorded in the foetuses: litter size, body weight, sex, anogenital distance of all live pups, external examination, soft tissue examination, head examination and skeletal examination.

Salivation was observed in 0/22, 1/22, 12/22 and 22/22 females in the control, low- mid- and high-dose group, respectively, in increasing frequency with increasing dose level. The salivation was not considered toxicologically relevant and was considered a physiological response following test substance administration rather than a sign of systemic toxicity.

In the high-dose group, the terminal body weight was 4% lower than control values (not significant). The mean gravid uterus weight was 7% lower than control (not statistically significant). As these differences were small and not statistically significant, they were not considered as adverse and not as toxicologically relevant.

The food consumption was reduced during the complete dosing period in high-dose females, compared with the control. During gestation day 6 – 21 the mean overall food intake was 10% lower, compared to the control. The reduced food consumption is not considered to be toxicologically relevant, as it was limited and did not affect the body weight.

Triiodothyronine (T3) levels were statistically significantly decreased in mid- and high-dose females (0.85 x and 0.75 x, respectively), compared with control. All the mean values fell within the historical control data range. No change were noted in the T4 and TSH levels. As the T3 values seemed to be affected in a dose-related manner, a treatment-related effect cannot be excluded. However, as no associate effects were noted and the T4 and TSH levels were similar in all groups, the changes were considered non-adverse.

The mean post-implantation loss was slightly higher in treatment groups compared with the control: 3.90, 7.54, 6.51 and 6.15% in the control, low- mid- and high-dose group, respectively. The mean values fell withinthe historical control data range and the difference wasnot statistically significant. This is not considered treatment-related.

No treatment-related effects were noted on mortality/moribundity, thyroid weight, macroscopic examination, litter loss, number of corpora lutea, number of pre-implantations, number of early and late resorptions, and number of live and dead fetuses.

The maternal NOAEL systemic was set at = 1000 mg/kg bw/day.

External malformations were observed in one fetus in the control group and in one fetus in the low-dose group. One fetus in the control group had a visceral malformation. Variations were noted in all the groups in single or small numbers. All these cases were considered incidental findings due to the single or low occurrence. There were no treatment-related skeletal malformations or variations. No treatment-related effects were noted on litter size, offspring body weight, sex ratio and anogenital distance.

The NOAEL developmental is 1000 mg/kg bw/day and the NOAEL teratogenicity is 1000 mg/kg bw/day.

 

Alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) was administered to female New Zealand White rabbits in a Prenatal developmental toxicity study according to OECD guideline 414 under GLP conditions (Shell, 2022b). Groups of 22 dams were administered doses of 100, 250 and 400 mg/kg bw/day by oral gavage, 7 days a week on gestation day 7 - 28. The control group was treated according to the same protocol and received the vehicle (propylene glycol) only. All the females were time-mated and the day of mating was gestation day 0 (Day 0 post-coitum). The dams were sacrificed on gestation day 29.

The following parameters were recorded in the dams: mortality/moribundity, clinical signs, body weight and food consumption, water consumption (monitored by visual inspection), macroscopic examination, gravid uterus weight, number of corpora lutea, number of implantations, number of early and late resorptions, and number of live and dead fetuses. The following parameters were recorded in the foetuses: body weight, sex, external examination, soft tissue examination, head examination and skeletal examination.

Females in all groups were sacrificed prematurely: 3/22, 2/22, 3/22 and 3/22 females in the control, low- mid- and high-dose group, respectively. Seven of these 11 deaths were considered treatment-related (1 in the low-dose group, 3 in the mid-dose group, and 3 in the high-dose group) and a summary of the observed effects is given below.

The animal in the low-dose group was sacrificed in extremis for animal welfare reasons on gestation day 17. The animal had limited to no food consumption for 7 consecutive days and slight body weight loss (4% compared to the highest recorded body weight on site). The animal did not show any clinical signs and no abnormal findings were noted at necropsy. Although a relationship to treatment with the test item could not be excluded, since mortality due to prolonged absent food consumption was also noted in one female of the control group and no effects on food consumption were noted in the remaining animals at 100 mg/kg/day, this death was not considered toxicologically relevant.

The three animals in the mid-dose group were sacrificed in extremis on gestation day 17, 24 and 20, respectively. Two of the animals had limited to no food consumption for 7 consecutive days and showed slight to moderate body weight loss (3 and 5%, respectively, compared to the highest recorded body weight on site). The third animal had severe body weight loss (11% lower than the highest recorded body weight). The food consumption was lower than for the control group but stable with the exception of gestation day 12-16 and 21-24. It was thin from gestation day 20 and erected fur was noted on gestation day 23 and 24. Decreased activity and/or lying on side were noted for this animal on gestation day 21, 23 and 24. At necropsy, gelatinous watery content of the stomach was noted, as well as a small thymus.

In the high-dose group, three animals were sacrificed prematurely on gestation day 20, 17 and 19 after limited to no food consumption for = 7 consecutive days. They had slight to moderate body weight loss (1 - 6% compared to the highest recorded body weight on site). At necropsy, no abnormalities were noted for these animals. The toxicological relevance of the morbidity occurring in animals in the mid- and high-dose groups could not be excluded.

Of the four non-treatment-related unscheduled sacrifices, 2 were related to gavage problems (1 control animal, 1 low-dose animal). The control animal was found dead on gestation day 22. Red discharge from the nose and mouth was noted shortly prior to its death, as well as laboured breathing and vocalizations. At necropsy, dark brown foci were noted on the caudal lobe of the right lung. The animal of the low-dose group was sacrificed in extremis for animal welfare reasons on gestation day 22, as it had laboured breathing and pale skin immediately after dosing and blood was noted on the gavage tube. At necropsy, this animal was found to have dark red foci on - as well as a perforation of - the caudal lobe of the right lung.

In the control group, one female was sacrificed in extremis on gestation day 11, as it had not eaten for 8 consecutive days and lost 7% of its body weight (compared to the highest recorded body weight on site). One control female was sacrificed in extremis on gestation day 19, as the animal had limited to no food consumption for 8 consecutive days and showed a body weight loss of 6%. No macroscopic abnormalities were noted at necropsy.

All the animals that were sacrificed prematurely were pregnant with normal implantations for the duration of the pregnancy. 

In the low-dose group, decreased activity was noted in 2/19 animals on individual or multiple days during gestation day 19 – 27 and 1/19 showed uncoordinated movement on gestation day 27. Laboured breathing was noted in 2/19 females gestation day 19 – 22.

Decreased activity was noted in the mid-dose group in 10/19 animals on individual or multiple days during gestation day21 – 28, while 1/19 was observed lying on its side on gestation day 23.

In the high-dose group, 2/19 females showed a combination of the following clinical signs on gestation day 20 and 22, respectively: severely uncoordinated movements with extensor rigidity of both hind limbs, rolling and/or shallow breathing, decreased activity, limited use (moderate) of
both hind limbs, severe lameness in both hind limbs, abnormal gate, lying on one side, difficulty
rising and/or abnormal respiratory rate. Moreover, extensor rigidity of both hind limbs, lying on the side, prostrate position, erected fur and/or hunched posture was also noted in 5/19 females on single occasions during gestation day 23 – 28.The clinical signs were considered toxicologically relevant in the mid- and high-dose group.

In the mid-dose group, slightly lower body weight gain was observed over gestation day 9 - 12 and 15 - 18 (not statistically significant). Overall body weight gain, body weight at the end of the dosing period and body weight gain corrected for gravid uterus weight were comparable to control. The effect was not considered adverse. In the high-dose group the overall mean body weight gain (gestation day7 – 29, measured in grams gained) was decreased by 35% compared to the control group (not statistically significant). Body weight gain corrected for gravid uterus weight was slightly lower compared to control (13%) but not statistically significant. The body weight changes were considered toxicologically relevant in the high-dose group.

The mean food consumption for gestation day 7 - 29 was reduced by 18% in the high-dose group, compared with the control. This was considered to be toxicologically relevant. As the food consumption in the low- and mid-dose groups was 6 - 7% lower than control and not statistically significant, this was not seen as adverse.  

In the females sacrificed according to the protocol on gestation day 29, dark foci of the stomach was noted in 1/19, 4/20, 2/19 and 1/19 females in the control, low- mid- and high-dose group, respectively. These changes were not considered treatment-related.

A slightly lower number of corpora lutea and implantations in the high-dose group was not treatment-related, as dosing with test item started after implantation on gestation day 7.

No treatment-related effects were noted on litter loss, number of pre- and post-implantations, number of early and late resorptions, and number of live and dead fetuses.

The maternal NOAEL systemic was set at 100 mg/kg bw/day, based on the morbidity and clinical signs at the highest dose level of 250 mg/kg bw/day.

In the offspring, the number of live fetuses in the high-dose group was lower than in the control (7.7 vs 8.6 in control, not statically significant). This was considered a consequence of the lower number of corpora lutea and hence the lower number of implantation sites at this dose, and was therefore not treatment-related.

No treatment-related effects were noted on litter size, body weight and sex ratio. There were no treatment-related external, skeletal or visceral malformations or variations.

The NOAEL developmental is 400 mg/kg bw/day and the NOAEL teratogenicity is 400 mg/kg bw/day.

 

Dose range-finding study

A dose range-finder study in pregnant rabbits was performed with alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) (20288391, Shell, 2021). In this study, 6 pregnant rabbits/group were exposed to the test substance via oral gavage at dose levels of 250, 500, and 750 mg/kg bw/day, 7 days a week on gestation day 7 - 28. In total, 7 animals (1 at 500 mg/kg bw/day, and 6 at 750 mg/kg bw/day) were sacrificed in extremis prior to the scheduled sacrifice.

One mid-dose animal was sacrificed in extremis on gestation day 19 as it had limited to no food consumption for 7 consecutive days and it had lost 9% of its weight compared to the highest body weight measured.

In the high-dose group, all animals were sacrificed before scheduled necropsy. Three of six (3/6) animals were sacrificed in extremis for animal welfare reasons (one animal gestation day 9 and 2 animals on gestation day 16). All three animals showed some type of episode (decreased activity, increased respiratory rate and extensor rigidity of fore- and hindlimbs, partly closed eyes, ataxia and moderately uncoordinated). Moreover, all three animals showed decreased food consumption and body weight loss.

One of six females (1/6) in the high-dose group was sacrificed in extremis for animal welfare reasons on gestation day 9. During the oral gavage procedure, red fluid on the dosing tube was noted. Laboured breathing, abnormal breathing sounds and shallow breathing as well as pallor skin was also noted and the animal was sacrificed. At necropsy, red watery fluid in the thoracic cavity was noted, as well as dark red discoloration of the right lung. This death was considered related to the gavage procedure and not to treatment.

The 2 remaining animals of this group were sacrificed on gestation day 16. For one animal, erected fur was noted gestation day 14 - 16, as well as decreased activity on gestation day 16. Food consumption was reduced for these animals from the start of the treatment onwards. Slight body weight loss was noted for these animals during gestation day 7 – 9 and 9 - 12, which appeared to stabilize to slight body weight gain gestation day 12 -15. At necropsy, no toxicologically relevant macroscopic findings were noted for these animals.

No treatment-related effects were noted on litter loss, number of corpora lutea, number of pre-implantations, and number of early and late resorptions. In the high-dose group the post-implantation loss was higher (13%) than in control (7%).

The number of viable and dead fetuses and fetal body weights were considered normal. The external examination of the fetuses did not show any abnormalities.

Based on the adverse findings and in consultation with the sponsor, the following dose levels were selected for the main study: 100, 250 and 400 mg/kg bw/day.

 

Studies in the AE category

Studies investigating toxicity to reproduction and developmental toxicity are available for the following AE substances (Table 1 - 3):

Table 1: Overview of OECD 422 studies, developmental toxicity

CAS No.	EC No.	Substance	Screening study (OECD 422)
			NOAEL developmental (F1) [mg/kg bw/day]	NOAEL systemic (F0) [mg/kg bw/day]
Linear subgroup
26183-52-8	500-046-6	Decan-1-ol, ethoxylated	= 950	= 950
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	= 1000	= 1000
9004-95-9	939-518-5	Hexadecan-1-ol, ethoxylated	= 1000	= 1000 (local: 300)
68439-49-6	939-518-5	Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO	= 1000	= 1000
9004-98-2	500-016-2	(Z)-9-Octadecen-1-ol ethoxylated	= 1000	= 1000
Mixed branched & linear subgroup
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	300	300
160901-19-9	500-457-0	Alcohols, C12-13, branched and linear, ethoxylated	300	= 1000
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	300	= 1000

Table 2: Overview of OECD 414 studies in the rat

CAS No.	EC No.	Substance	Prenatal developmental toxicity study (OECD 414) in the rat
			NOAEL [mg/kg bw/day]
			Systemic (maternal)	Development	Teratogenicity
Linear
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	300	300	= 1000
68920-66-1	500-236-9	Alcohols, C16-18 and C18-unsatd., ethoxylated	1000	1000	1000
Mixed linear & branched
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	800	800	800
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	1000	1000	1000

 

Table 3: Overview of OECD 414 studies in the rabbit

CAS No.	EC No.	Substance	Prenatal developmental toxicity study (OECD 414) in the rabbit
			NOAEL [mg/kg bw/day]
			Systemic (F0)	Development (F1)	Teratogenicity (F1)
Linear
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	30	200	200
68920-66-1	500-236-9	Alcohols, C16-18 and C18-unsatd., ethoxylated	(study ongoing)	-	-
Mixed linear & branched
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	100	400	400

Conclusion on developmental toxicity

The data on developmental toxicity available for alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) is mainly consistent with the overall toxicity to reproduction data for AE substances. In the OECD 422 study with alcohols, C12-15, branched and linear, ethoxylated, the mean body weight of male pups was statistically significantly decreased at PND 7 and 13 in the high-dose group (1000 mg/kg bw/day). In addition, mean body weights of female pups in the high-dose group were decreased at PND 13, though not reaching statistical significance. These findings were considered adverse. However, a significant result was only observed in males at PND 7 and 13 and not in the combined mean body weight for males and females, or for females individually. The reduced weight of male pups is therefore more likely an incidental effect as it is unlikely males put on less weight than females under the same treatment circumstances. No effects on body weight were recorded in the prenatal developmental toxicity study performed with alcohols, C12-15, branched and linear, ethoxylated. No effect on development or teratogenicity was noted in the OECD 414 studies in the rat and the rabbit.

The following NOAELs were set:

Oral (rat, OECD 414): NOAEL (developmental toxicity) = 1000 mg/kg bw/day

Oral (rat, OECD 414): NOAEL (teratogenicity) = 1000 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (developmental toxicity) = 400 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (teratogenicity) = 400 mg/kg bw/day

 

For a detailed evaluation of the toxicity to reproduction potential of the substances in the AE category, please refer to the category justification attached to the category object.

Justification for classification or non-classification

The available data on toxicity to reproduction obtained with alcohols, C12-15, branched and linear, ethoxylated (CAS 106232-83-1 / EC 500-294-5) and with other members of the Alcohol Ethoxylates (AE) category do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.