Benzoic acid, 2-hydroxy-, C14-18-alkyl derivs.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2015 to 06 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Age at study initiation: (P) Approximately 11 to 12 weeks
- Weight at study initiation (mean): (P) Males: 308 g; Females: 222 g
- Fasting period before study: No
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis (plastic cages, height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During the lactation period, pups were kept with the dam until termination. Sterilised sawdust was used as bedding material and paper as cage-enrichment/nesting material was supplied.
- Diet: ad libitum pelleted rodent diet
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 09 February 2015 (males); 23, 24 and 26 February 2015 and 02 and 06 March 2015 (females and pups)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stored during the dosing of all animals. Adjustment was made for specific gravity of the vehicle and the test material.

DOSE VOLUME: 5 mL/kg bw

VEHICLE
- Specific gravity: 1.036

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating; after this, females who had not shown evidence of mating were separated from their males.
- Proof of pregnancy: 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.
- After successful mating each pregnant female was caged (how): Once mating occurred, the males and females were separated. Females were individually housed in plastic cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Samples of dose preparations were taken on a single occasion and were stored and dispatched on dry ice to the test site for formulation analysis.
Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85 to 115 % of target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

SAMPLES
In total, 16 samples were included in this study, distributed over 4 formulation groups. The samples of the control group and the 50 mg/kg dose group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 15 and 150 mg/kg were taken in duplicate from the top, middle and bottom position of the container.
The samples were stored at a target temperature =-70 °C.

ANALYTICAL METHOD
- Analytical conditions
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections
The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations was acquired in the following order: analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples and procedural recovery samples high and low (replicate 2).
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAS
Response (Y): Peak area test material [mAU]

Calibration curve: Y = a + bX + cX²
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept

Analysed concentration (X): X = [-b + v(b² - 4c(a – y)) / 2c] x ((V x d) / w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
[-b + v(b² - 4c(a – y)) / 2c] values are obtained from Analyst® version 1.5.2 (Applied Biosystems, Burlington, Canada).

Recovery: (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy: (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.): ((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
- Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted. Outliers could be discarded as long as minimally 75 % of the responses of the calibration were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 85 to 115 %.
The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 85 to 115 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

RESULTS
- Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a (1 / concentration²) weighting factor. The coefficient of correlation (r) was 1.0000.
No response at the retention time of the test material in the analytical blank samples was detected.
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 85 to 115 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the dose groups were in agreement with the target concentrations (i.e. mean accuracies between 85 and 115 %).
The formulations of the 15 and 150 mg/kg dose groups were homogeneous (i.e. coefficient of variation = 10 %).

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Female nos. 41, 43, 45 (control), 52, 55, 56 (15 mg/kg) and 74 (150 mg/kg) were not dosed on Day 1 of lactation as these females were littering at the time of dosing.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 15, 50 and 150 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 28-day study with the test material, in which dosages of 30, 100 and 300 mg/kg/day were administered by daily oral gavage.
No mortality occurred. Incidental cases of hunched posture and piloerection occurred in the high dose. Males at 300 mg/kg/day showed a lower body weight gain with slightly lower food intake throughout treatment, but body weight gain and food intake of females at 300 mg/kg/day was considered unaffected by treatment.
Haematological changes were confined to a higher white blood cell count in males and females at 300 mg/kg/day (and lower relative eosinophil count in these males). Clinical biochemistry changes at 300 mg/kg/day included higher alanine and aspartate aminotransferase and alkaline phosphatase activities in both sexes, lower total protein levels in males, and higher potassium and inorganic phosphate and lower chloride levels in females.
Necropsy showed an irregular surface of the forestomach in all males and females at 300 mg/kg/day.
A higher relative liver weight was recorded for males and females at 300 mg/kg/day, and for females also at 100 mg/kg/day (relative weight was approximately 17 and 23 % higher than controls for males and females at 300 mg/kg/day, respectively. At 100 mg/kg/day, the relative liver weight increase was approximately 9 %). Other organ weight changes in males were attributed to lower terminal body weights.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily animals were examined for mortality/viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals at least immediately (0 to 30 minutes) and between 1 and 2 hours (± 30 minutes) after dosing. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: GENERAL REPRODUCTION DATA
- Parameters observed: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Sperm parameters (parental animals):

Parameters examined in [all] male parental animals: testis weight and epididymis weight and staging of spermatogenesis.

Litter observations:

PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.
-Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated.
- Male animals: Males were necropsied following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: Females which delivered were necropsied on lactation Days 5 to 7. Females which failed to deliver (nos. 59 and 63) were necropsied on post-coitum Days 26 and 21 (females with and without evidence of mating, respectively). A female with total litter loss (no. 43) was necropsied within 24 hours of litter loss.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution): cervix, clitoral gland, coagulation gland, epididymides, female mammary gland area, liver, ovaries, preputial gland, prostate gland, seminal vesicles, stomach, testes, uterus, vagina and all gross lesions.
The epididymides and testes were fixed in modified Davidson's solution and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Testes and epididymides weight and terminal body weight was recorded for all parental males on the scheduled day of necropsy.

All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 µm. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.

The following slides were examined by a pathologist:
- Ovaries, testes, epididymides, stomach and liver of the animals of the control and 150 mg/kg dose groups.
- Stomach of all 15 and 50 mg/kg dose group animals based on (possible) treatment-related changes in this organ in the high dose group.
- The additional slides of the testes of the males of the control and 150 mg/kg dose groups and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups, i.e. female/male numbers 43/3 (control group), 59/19 (15 mg/kg dose group) and 63/23 (50 mg/kg dose group). Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5 to 7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. Also if possible, defects or cause of death were determined.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data. Test statistics were calculated on the basis of exact values for means and pooled variances.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = (Number of pregnant females / Number of paired females) x 100
Conception index (%) = (Number of pregnant females / Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

For each group, the following calculations were performed:
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
Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at first litter check) x 100
Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

MORTALITY (PARENTAL ANIMALS)
No treatment-related mortality occurred during the study period.
One control female (no.43) was sacrificed due to total litter loss on post-coitum Day 24. Since this occurred in the control group, this was not related to treatment with the test material.

CLINICAL SIGNS (PARENTAL ANIMALS)
No clinical signs were noted during the observation period that were considered to be toxicologically relevant.
Piloerection shown by one female at 150 mg/kg/day was considered not toxicologically relevant since this finding occurred incidentally and was absent among other animals of this dose group.
Salivation seen after dosing among animals at 50 and 150 mg/kg/day during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity, considering the nature and minor severity of the effect. This sign may be related to irritancy/taste of the test material.
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
Males at 150 mg/kg/day had statistically significantly lower body weight and body weight gain on Day 8 of the premating period, which appeared lower throughout the remainder of the treatment period (but not statistically significant). The mean body weight of these males was reduced by approximately 5 or 6 % compared to the control on the last day of treatment and at necropsy, respectively.
Body weights and body weight gain of other treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Food intake (before and after correction for body weight) of males at 150 mg/kg/day appeared marginally lower than controls over Days 1 to 8 of the premating period.
The statistically significantly lower food consumption of females at 150 mg/kg/day between Days 11 to 14 of the post-coitum period was considered not toxicologically relevant, since this slight change was only temporarily seen over the entire treatment period.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Test material-related irregular surface was observed in the forestomach of 1/10 males treated at 50 mg/kg/day and in 3/10 males and 1/10 females treated at 150 mg/kg/day.
The other recorded macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test material-related alterations in testes and epididymides weights.
The significant increase in organ to body weight ratio of the testes at 150 mg/kg/day was considered not test material-related since this was not accompanied by a significant increase in absolute testes weight. This was considered to be due to the slight decrease in body weight recorded for the males at 150 mg/kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test material-related microscopic findings were noted in the forestomach of males and females treated at 50 and 150 mg/kg/day and consisted of:
- Granulocytic subepithelial inflammation in 8/10 males (8 minimal) and 4/10 females (4 minimal) at 50 mg/kg/day and in 10/10 males (2 minimal, 8 slight) and 9/10 females (5 minimal, 4 slight) at 150 mg/kg/day.
- Hyperkeratosis, diffuse in 10/10 males (8 minimal, 2 slight) and 5/10 females (5 minimal) at 50 mg/kg/day and in 10/10 males (8 slight, 2 moderate) and 10/10 females (3 minimal, 6 slight, 1 moderate) at 150 mg/kg/day.
- Submucosal oedema in 1/10 males (slight) and 1/10 females (minimal) at 150 mg/kg/day.
- Erosion in 1/10 males (minimal) at 150 mg/kg/day.
There were no other test material-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were two pairs, one of the 15 mg/kg dose group (male 19, female 59) and one of the 50 mg/kg dose group (male 23, female 63) that failed to sire or deliver pups. No cause for the failure to sire or deliver offspring could be established from the sections examined. In addition, the female of one control group pair (male 3 and female 43) was sacrificed after total litter loss. For female 43, the moderate lobar necrosis of the liver together with the macroscopically seen ripped placenta were the likely cause for the total litter loss.
Spermatogenic staging profiles were normal for all males examined.

REPRODUCTION DATA (PARENTAL ANIMALS)
Reproductive parameters (mating, fertility and conception indices, pre-coital time, and number of corpora lutea and implantation sites) were considered to have been unaffected by treatment.
One female at 50 mg/kg/day (no. 63) showed no evidence of mating. One female at 15 mg/kg/day (no. 59) had implantation sites only. The incidence of these occurrences remained within the range to be expected for rats of this age and strain, and showed no dose-related trend.
For control female nos. 44, 45, 49 and 50, and a single female each at 15 and 50 mg/kg/day (nos. 53 and 68, respectively), the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered caused by normal resorption of these areas as these enumerations were performed between Days 5 and 7 of lactation.

PARTURITION/MATERNAL CARE (PARENTAL ANIMALS)
One control female (no. 43) had total litter loss on post-coitum Day 24. This female showed evidence of delivery on the preceding day. On the day of sacrifice, no pups were found in the cage and no pups resided in the uterus based on palpation. Since this total litter loss occurred in the control group, this was unrelated to treatment with the test material.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
No other treatment-related or toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, parturition or maternal care.

DISCUSSION AND CONCLUSION
At 50 and 150 mg/kg/day, local forestomach lesions were noted in both sexes which consisted of hyperkeratosis (up to moderate degree, correlating to irregular surface of the forestomach seen macroscopically), subepithelial granulocytic inflammation (up to slight degree), submucosal oedema (up to slight degree) and/or erosion (minimal degree in one male at 150 mg/kg/day). The hyperkeratosis, submucosal oedema, erosion and the resulting inflammatory response (granulocytic subepithelial inflammation) in the forestomach are likely the result of an irritating/damaging effect of the test material.
As such, these findings were considered adverse in nature.
At 150 mg/kg/day, the slightly lower body weight and body weight gain of males recorded from Day 8 of the premating period onwards and slightly lower food intake during the first week of the premating period was determined to be not adverse, given that these changes were considered mild in nature. It could not be excluded that this slight change in body weight and food intake was related to the aforementioned forestomach lesions.
No toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. clinical appearance, food consumption and microscopic examination).
No reproductive toxicity was observed up to the highest dose level tested (150 mg/kg/day).
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites, testes and epididymides weights, staging of spermatogenesis and histopathological examination of reproductive organs).

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the presence of adverse forestomach lesions starting at 50 mg/kg/day in both sexes.

Dose descriptor:

NOAEL

Remarks:

- systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL was at least 150 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

- reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL was at least 150 mg/kg/day.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

DEVELOPMENTAL DATA (OFFSPRING)
At 150 mg/kg/day, body weight of male pups (and for pups of both sexes combined) was increased attaining statistical significance on Day 1 of lactation.
Other markers for early postnatal pup development (mortality, clinical signs and macroscopy) and gestation index and duration, parturition, maternal care were considered to have been unaffected by treatment.

EARLY POSTNATAL PUP DEVELOPMENT (OFFSPRING)
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment. Clinical signs and external macroscopy did not reveal any treatment-related findings.

MORTALITY (OFFSPRING)
Six pups of the control group and one pup at 50 mg/kg/day were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Dehydrated appearance was noted for one surviving pup at 15 mg/kg/day, the nature and incidence of which remained within the range considered normal for pups of this age. This sign was therefore considered to be of no toxicological relevance.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings for pups that were found dead included autolysis and absence of milk in the stomach. Dehydrated appearance was noted for one surviving pup at 15 mg/kg/day. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Table 1: Summary of Reproduction data

	Dose Group (mg/kg)
	0	15	50	150
Females paired	10	10	10	10
Females mated	10	10	9	10
Females pregnant	10	9	9	10
Females with living pups on Day 1	9	9	9	10
Mating index (%)	100	100	90	100
Fertility index (%)	100	90	90	100
Conception index (%)	100	90	100	100
Gestation index (%)	90	100	100	100

One female form the 15 mg/kg dose group had implantation sites only. One female from the control group exhibited total litter loss.

Table 2: Number of Females Mated on Each Day During the Pairing Period

Day of the Pairing Period	Dose Group (mg/kg)
	0	15	50	150
1	3	-	4	2
2	4	4	3	3
3	1	4	1	3
4	1	2	-	2
14	1	-	1	-
Median pre-coital time (days)	2	3	2	3
Mean pre-coital time (days)	3.2	2.8	3.0	2.5
N	10	10	9	10

 

Table 3: Summary of Corpora Lutea and Implantation Sites

Parameter		Dose Group (mg/kg)
		0	15	50	150
Corpora lutea	Mean SD N	13.6 3.2 10	14.1 4.5 10	13.9 1.5 9	13.0 2.5 10
Implantations	Mean SD N	10.9 4.6 10	12.7 4.6 10	13.2 1.7 9	11.8 2.8 10

SD = standard deviation

Conclusions:

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 15 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 150 mg/kg/day was derived.

Executive summary:

The potential of the test material to cause reproductive toxicity was investigated in a reproduction/developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 421 and US EPA OPPTS 870.3550 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats at dose levels of 0, 15, 50 and 150 mg/kg/day. Each group consisted of 10 males and 10 females. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs, body weight and food consumption, macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, pre-coital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity. The analysis showed that the formulations were prepared accurately and were homogenous.

Parental results:

At 50 and 150 mg/kg/day, local forestomach lesions were noted in both sexes which consisted of hyperkeratosis (correlating to irregular surface of the forestomach seen macroscopically), subepithelial granulocytic inflammation, submucosal oedema and/or erosion. The hyperkeratosis, submucosal oedema, erosion and the resulting inflammatory response (granulocytic subepithelial inflammation) in the forestomach are likely the result of an irritating/damaging effect of the test material. As such, these findings were considered adverse in nature.

At 150 mg/kg/day, the slightly lower body weight and body weight gain of males recorded from Day 8 of the premating period onwards and slightly lower food intake during the first week of the premating period was determined to be not adverse, given that these changes were considered mild in nature. It could not be excluded that this slight change in body weight and food intake was related to the aforementioned forestomach lesions.

No toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. clinical appearance, food consumption and microscopic examination).

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites, testes and epididymides weights, staging of spermatogenesis and histopathological examination of reproductive organs).

Developmental results:

No adverse reproductive findings were observed up to the highest dose level tested (150 mg/kg/day).

At 150 mg/kg/day, body weight of male pups (and for pups of both sexes combined) was increased on Day 1 of lactation. This change was considered to be not adverse in nature, since this change was slight and temporary in nature (i.e. on Day 4 of lactation body weights were similar to controls), and since there were no other treatment-related changes in developmental parameters.

No other treatment-related or toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 15 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 150 mg/kg/day was derived.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted in accordance with standardised guidelines under GLP conditions. It was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be good.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The potential of the test material to cause reproductive toxicity was investigated in a reproduction/developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 421 and US EPA OPPTS 870.3550 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats at dose levels of 0, 15, 50 and 150 mg/kg/day. Each group consisted of 10 males and 10 females. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs, body weight and food consumption, macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, pre-coital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity. The analysis showed that the formulations were prepared accurately and were homogenous.

Parental results:

At 50 and 150 mg/kg/day, local forestomach lesions were noted in both sexes which consisted of hyperkeratosis (correlating to irregular surface of the forestomach seen macroscopically), subepithelial granulocytic inflammation, submucosal oedema and/or erosion. The hyperkeratosis, submucosal oedema, erosion and the resulting inflammatory response (granulocytic subepithelial inflammation) in the forestomach are likely the result of an irritating/damaging effect of the test material. As such, these findings were considered adverse in nature.

At 150 mg/kg/day, the slightly lower body weight and body weight gain of males recorded from Day 8 of the premating period onwards and slightly lower food intake during the first week of the premating period was determined to be not adverse, given that these changes were considered mild in nature. It could not be excluded that this slight change in body weight and food intake was related to the aforementioned forestomach lesions.

No toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. clinical appearance, food consumption and microscopic examination).

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites, testes and epididymides weights, staging of spermatogenesis and histopathological examination of reproductive organs).

Developmental results:

No adverse reproductive findings were observed up to the highest dose level tested (150 mg/kg/day).

At 150 mg/kg/day, body weight of male pups (and for pups of both sexes combined) was increased on Day 1 of lactation. This change was considered to be not adverse in nature, since this change was slight and temporary in nature (i.e. on Day 4 of lactation body weights were similar to controls), and since there were no other treatment-related changes in developmental parameters.

No other treatment-related or toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 15 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 150 mg/kg/day was derived.

 

EOGRTS

In accordance with Annex XI, Section 3, the Extended One-Generation Reproductive Toxicity Study (EOGRTS) may be omitted based on exposure scenarios developed in the Chemical Safety Report, given that the comparison of the derived DNEL with the results of the exposure assessment indicate that exposures are always well below the derived DNEL.

 

Although, Annex XI, Section 3 (June 1, 2015), states that “a DNEL derived from a screening test for reproductive/developmental toxicity shall not be considered appropriate to omit a prenatal developmental toxicity or a two-generation reproductive toxicity study”, paragraph 5 to Annexes IX and X states that “When, for certain endpoints, information is not provided for other reasons than those mentioned in Column 2 of this Annex or in Annex XI, this fact and the reasons shall also be clearly stated”

 

Therefore, as demonstrated in the CSR, the very low potential for exposure to the substance throughout its lifecycle due to its manufacture and use taking place in a rigorously contained system with emission minimisation controls, coupled with the risk characterisation ratios being significantly less than 1, are considered sufficient grounds to omit this study. Any potential extra information that would be gained from the performance of this test are outweighed by the opportunity to prevent unnecessary vertebrate animal testing.

 

The Registrant therefore proposes to waive the study required to address this endpoint.

Short description of key information:
ORAL
NOAEL 150 mg/kg/day (maternal systemic and reproductive toxicity), rat, OECD 421, US EPA OPPTS 870.3550

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information

ORAL
NOAEL 150 mg/kg/day (maternal systemic and developmental toxicity), rat, OECD 421, US EPA OPPTS 870.3550

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2015 to 06 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Age at study initiation: (P) Approximately 11 to 12 weeks
- Weight at study initiation (mean): (P) Females: 222 g
- Fasting period before study: No
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis (plastic cages, height 18 cm). Post-mating, females were individually housed in plastic cages (height 18 cm). During the lactation period, pups were kept with the dam until termination. Sterilised sawdust was used as bedding material and paper as cage-enrichment/nesting material was supplied.
- Diet: ad libitum pelleted rodent diet
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 23, 24 and 26 February 2015 and 02 and 06 March 2015 (females and pups)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stored during the dosing of all animals. Adjustment was made for specific gravity of the vehicle and the test material.

DOSE VOLUME: 5 mL/kg bw

VEHICLE
- Specific gravity: 1.036

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Samples of dose preparations were taken on a single occasion and were stored and dispatched on dry ice to the test site for formulation analysis.
Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85 to 115 % of target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

SAMPLES
In total, 16 samples were included in this study, distributed over 4 formulation groups. The samples of the control group and the 50 mg/kg dose group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 15 and 150 mg/kg were taken in duplicate from the top, middle and bottom position of the container.
The samples were stored at a target temperature =-70 °C.

ANALYTICAL METHOD
- Analytical conditions
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections
The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations was acquired in the following order: analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples and procedural recovery samples high and low (replicate 2).
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAS
Response (Y): Peak area test material [mAU]

Calibration curve: Y = a + bX + cX²
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept

Analysed concentration (X): X = [-b + v(b² - 4c(a – y)) / 2c] x ((V x d) / w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
[-b + v(b² - 4c(a – y)) / 2c] values are obtained from Analyst® version 1.5.2 (Applied Biosystems, Burlington, Canada).

Recovery: (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy: (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.): ((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
- Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted. Outliers could be discarded as long as minimally 75 % of the responses of the calibration were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 85 to 115 %.
The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 85 to 115 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

RESULTS
- Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a (1 / concentration²) weighting factor. The coefficient of correlation (r) was 1.0000.
No response at the retention time of the test material in the analytical blank samples was detected.
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 85 to 115 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the dose groups were in agreement with the target concentrations (i.e. mean accuracies between 85 and 115 %).
The formulations of the 15 and 150 mg/kg dose groups were homogeneous (i.e. coefficient of variation = 10 %).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating; after this, females who had not shown evidence of mating were separated from their males.
- Proof of pregnancy: 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.
- After successful mating each pregnant female was caged (how): Once mating occurred, the males and females were separated. Females were individually housed in plastic cages.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Female nos. 41, 43, 45 (control), 52, 55, 56 (15 mg/kg) and 74 (150 mg/kg) were not dosed on Day 1 of lactation as these females were littering at the time of dosing.

Frequency of treatment:

Once daily

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 28-day study with the test material, in which dosages of 30, 100 and 300 mg/kg/day were administered by daily oral gavage.
No mortality occurred. Incidental cases of hunched posture and piloerection occurred in the high dose. Males at 300 mg/kg/day showed a lower body weight gain with slightly lower food intake throughout treatment, but body weight gain and food intake of females at 300 mg/kg/day was considered unaffected by treatment.
Haematological changes were confined to a higher white blood cell count in males and females at 300 mg/kg/day (and lower relative eosinophil count in these males). Clinical biochemistry changes at 300 mg/kg/day included higher alanine and aspartate aminotransferase and alkaline phosphatase activities in both sexes, lower total protein levels in males, and higher potassium and inorganic phosphate and lower chloride levels in females.
Necropsy showed an irregular surface of the forestomach in all males and females at 300 mg/kg/day.
A higher relative liver weight was recorded for males and females at 300 mg/kg/day, and for females also at 100 mg/kg/day (relative weight was approximately 17 and 23 % higher than controls for males and females at 300 mg/kg/day, respectively. At 100 mg/kg/day, the relative liver weight increase was approximately 9 %). Other organ weight changes in males were attributed to lower terminal body weights.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily animals were examined for mortality/viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals at least immediately (0 to 30 minutes) and between 1 and 2 hours (± 30 minutes) after dosing. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: GENERAL REPRODUCTION DATA
- Parameters observed: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.


SACRIFICE
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated.
- Maternal animals: Females which delivered were necropsied on lactation Days 5 to 7. Females which failed to deliver (nos. 59 and 63) were necropsied on post-coitum Days 26 and 21 (females with and without evidence of mating, respectively). A female with total litter loss (no. 43) was necropsied within 24 hours of litter loss.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution): cervix, clitoral gland, coagulation gland, female mammary gland area, liver, ovaries, preputial gland, stomach, uterus, vagina and all gross lesions.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.

All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 µm. These slides were stained with haematoxylin and eosin.

The following slides were examined by a pathologist:
- Ovaries, stomach and liver of the animals of the control and 150 mg/kg dose groups.
- Stomach of all 15 and 50 mg/kg dose group animals based on (possible) treatment-related changes in this organ in the high dose group.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all females that failed to deliver healthy pups, i.e. female numbers 43 (control group), 59 (15 mg/kg dose group) and 63 (50 mg/kg dose group). Reproductive organs included the cervix, clitoral gland, coagulation gland, ovaries, preputial gland, uterus, and vagina.
An attempt was made to correlate gross observations with microscopic findings.

Ovaries and uterine content:

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Fetal examinations:

PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.
-Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5 to 7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. Also if possible, defects or cause of death were determined.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data. Test statistics were calculated on the basis of exact values for means and pooled variances.

Indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = (Number of pregnant females / Number of paired females) x 100
Conception index (%) = (Number of pregnant females / Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
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
Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at first litter check) x 100
Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY (PARENTAL ANIMALS)
No treatment-related mortality occurred during the study period.
One control female (no.43) was sacrificed due to total litter loss on post-coitum Day 24. Since this occurred in the control group, this was not related to treatment with the test material.

CLINICAL SIGNS (PARENTAL ANIMALS)
No clinical signs were noted during the observation period that were considered to be toxicologically relevant.
Piloerection shown by one female at 150 mg/kg/day was considered not toxicologically relevant since this finding occurred incidentally and was absent among other animals of this dose group.
Salivation seen after dosing among animals at 50 and 150 mg/kg/day during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity, considering the nature and minor severity of the effect. This sign may be related to irritancy/taste of the test material.
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
The statistically significantly lower food consumption of females at 150 mg/kg/day between Days 11 to 14 of the post-coitum period was considered not toxicologically relevant, since this slight change was only temporarily seen over the entire treatment period.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Test material-related irregular surface was observed in the forestomach of 1/10 females treated at 150 mg/kg/day.
The other recorded macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test material-related alterations in organ weights.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test material-related microscopic findings were noted in the forestomach of females treated at 50 and 150 mg/kg/day and consisted of:
- Granulocytic subepithelial inflammation in 4/10 females (4 minimal) at 50 mg/kg/day and 9/10 females (5 minimal, 4 slight) at 150 mg/kg/day.
- Hyperkeratosis, diffuse in 5/10 females (5 minimal) at 50 mg/kg/day and in 10/10 females (3 minimal, 6 slight, 1 moderate) at 150 mg/kg/day.
- Submucosal oedema in 1/10 females (minimal) at 150 mg/kg/day.
There were no other test material-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were two pairs, one of the 15 mg/kg dose group (male 19, female 59) and one of the 50 mg/kg dose group (male 23, female 63) that failed to sire or deliver pups. No cause for the failure to sire or deliver offspring could be established from the sections examined. In addition, the female of one control group pair (male 3 and female 43) was sacrificed after total litter loss. For female 43, the moderate lobar necrosis of the liver together with the macroscopically seen ripped placenta were the likely cause for the total litter loss.

REPRODUCTION DATA (PARENTAL ANIMALS)
Reproductive parameters (mating, fertility and conception indices, pre-coital time, and number of corpora lutea and implantation sites) were considered to have been unaffected by treatment.
One female at 50 mg/kg/day (no. 63) showed no evidence of mating. One female at 15 mg/kg/day (no. 59) had implantation sites only. The incidence of these occurrences remained within the range to be expected for rats of this age and strain, and showed no dose-related trend.
For control female nos. 44, 45, 49 and 50, and a single female each at 15 and 50 mg/kg/day (nos. 53 and 68, respectively), the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered caused by normal resorption of these areas as these enumerations were performed between Days 5 and 7 of lactation.

PARTURITION/MATERNAL CARE (PARENTAL ANIMALS)
One control female (no. 43) had total litter loss on post-coitum Day 24. This female showed evidence of delivery on the preceding day. On the day of sacrifice, no pups were found in the cage and no pups resided in the uterus based on palpation. Since this total litter loss occurred in the control group, this was unrelated to treatment with the test material.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

DISCUSSION AND CONCLUSION
At 50 and 150 mg/kg/day, local forestomach lesions were noted which consisted of hyperkeratosis (up to moderate degree, correlating to irregular surface of the forestomach seen macroscopically), subepithelial granulocytic inflammation (up to slight degree) and submucosal oedema (up to minimal degree). The hyperkeratosis, submucosal oedema, erosion and the resulting inflammatory response (granulocytic subepithelial inflammation) in the forestomach are likely the result of an irritating/damaging effect of the test material.
As such, these findings were considered adverse in nature.
No toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. clinical appearance, food consumption and microscopic examination).
No reproductive toxicity was observed up to the highest dose level tested (150 mg/kg/day).
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites, and histopathological examination of reproductive organs).

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

- systemic

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
DEVELOPMENTAL DATA (OFFSPRING)
At 150 mg/kg/day, body weight of male pups (and for pups of both sexes combined) was increased attaining statistical significance on Day 1 of lactation.
Other markers for early postnatal pup development (mortality, clinical signs and macroscopy) and gestation index and duration, parturition, maternal care were considered to have been unaffected by treatment.

EARLY POSTNATAL PUP DEVELOPMENT (OFFSPRING)
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment. Clinical signs and external macroscopy did not reveal any treatment-related findings.

MORTALITY (OFFSPRING)
Six pups of the control group and one pup at 50 mg/kg/day were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Dehydrated appearance was noted for one surviving pup at 15 mg/kg/day, the nature and incidence of which remained within the range considered normal for pups of this age. This sign was therefore considered to be of no toxicological relevance.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings for pups that were found dead included autolysis and absence of milk in the stomach. Dehydrated appearance was noted for one surviving pup at 15 mg/kg/day. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

not determinable due to absence of adverse toxic effects

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Table 1: Summary of Developmental data

Parameter	Dose Group (mg/kg)
	0	15	50	150
Total number of litters	9	9	9	10
Duration of gestation (days) Mean SD N	21.4 0.7 9	21.8 0.4 9	21.2 0.7 9	21.8 0.4 10
Dead pups at first litter check Number of litters affected Total Mean SD N	0 0 0.0 0.0 9	0 0 0.0 0.0 9	1 1 0.1 0.3 9	0 0 0.0 0.0 10
Living pups at first litter check Percentage of males / females Total Mean SD N	48 / 52 103 11.4 3.3 9	47 / 53 121 13.4 1.5 9	44 / 56 105 11.7 1.5 9	50 / 50 108 10.8 2.8 10
Postnatal loss Percentage of living pups Number of litters affected Total Mean SD N	5.8 2 6 0.7 1.7 9	0.0 0 0** 0.0 0.0 9	0.0 0 0* 0.0 0.0 9	0.0 0 0* 0.0 0.0 10
Viability index	94.2	100.0**	100.0*	100.0*

SD = Standard deviation

*Fisher's Exact test significant at the 5 % level

**Fisher's Exact test significant at the 1 % level 

Table 2: Summary of Pup Body Weights (g)

Lactation Day	Sex		Dose Group (mg/kg)
			0	15	50	150
1	M	Mean SD N	6.4 0.7 9	6.4 0.3 9	6.5 0.5 9	7.1* 0.5 10
	F	Mean SD N	6.1 0.7 9	6.2 0.5 9	6.1 0.6 9	6.6 0.4 10
	M + F	Mean SD N	6.2 0.7 9	6.3 0.4 9	6.2 0.6 9	6.9* 0.5 10
4	M	Mean SD N	9.5 1.5 9	9.5 0.6 9	9.3 0.8 9	10.1 1.2 10
	F	Mean SD N	9.2 1.5 9	9.0 0.6 9	8.8 0.9 9	9.5 1.1 10
	M + F	Mean SD N	9.4 1.5 9	9.2 0.6 9	9.0 0.9 9	9.8 1.1 10

SD = Standard deviation

*Significant at the 5 % level using the Dunnett-test based on pooled variance

Conclusions:

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 15 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 150 mg/kg/day was derived.

Executive summary:

The potential of the test material to cause developmental toxicity was investigated in a reproduction/developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 421 and US EPA OPPTS 870.3550 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats at dose levels of 0, 15, 50 and 150 mg/kg/day. Each group consisted of 10 males and 10 females. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs, body weight and food consumption, macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, pre-coital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity. The analysis showed that the formulations were prepared accurately and were homogenous.

Parental results:

At 50 and 150 mg/kg/day, local forestomach lesions were noted which consisted of hyperkeratosis (correlating to irregular surface of the forestomach seen macroscopically), subepithelial granulocytic inflammation, submucosal oedema and/or erosion. The hyperkeratosis, submucosal oedema, erosion and the resulting inflammatory response (granulocytic subepithelial inflammation) in the forestomach are likely the result of an irritating/damaging effect of the test material. As such, these findings were considered adverse in nature.

No toxicologically significant changes were noted in any of the other parental parameters investigated in this study.

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs).

Developmental results:

No adverse reproductive findings were observed up to the highest dose level tested (150 mg/kg/day).

At 150 mg/kg/day, body weight of male pups (and for pups of both sexes combined) was increased on Day 1 of lactation. This change was considered to be not adverse in nature, since this change was slight and temporary in nature (i.e. on Day 4 of lactation body weights were similar to controls), and since there were no other treatment-related changes in developmental parameters.

No other treatment-related or toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 15 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 150 mg/kg/day was derived.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted in accordance with standardised guidelines under GLP conditions. It was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be good.

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

The potential of the test material to cause developmental toxicity was investigated in a reproduction/developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 421 and US EPA OPPTS 870.3550 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats at dose levels of 0, 15, 50 and 150 mg/kg/day. Each group consisted of 10 males and 10 females. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs, body weight and food consumption, macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, pre-coital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity. The analysis showed that the formulations were prepared accurately and were homogenous.

Parental results:

At 50 and 150 mg/kg/day, local forestomach lesions were noted which consisted of hyperkeratosis (correlating to irregular surface of the forestomach seen macroscopically), subepithelial granulocytic inflammation, submucosal oedema and/or erosion. The hyperkeratosis, submucosal oedema, erosion and the resulting inflammatory response (granulocytic subepithelial inflammation) in the forestomach are likely the result of an irritating/damaging effect of the test material. As such, these findings were considered adverse in nature.

No toxicologically significant changes were noted in any of the other parental parameters investigated in this study.

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs).

Developmental results:

No adverse reproductive findings were observed up to the highest dose level tested (150 mg/kg/day).

At 150 mg/kg/day, body weight of male pups (and for pups of both sexes combined) was increased on Day 1 of lactation. This change was considered to be not adverse in nature, since this change was slight and temporary in nature (i.e. on Day 4 of lactation body weights were similar to controls), and since there were no other treatment-related changes in developmental parameters.

No other treatment-related or toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 15 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 150 mg/kg/day was derived.

 

OECD 414

In accordance with Annex XI, Section 3, the pre-natal developmental toxicity study required under Section 8.7.2 of the REACH Regulation may be omitted based on exposure scenarios developed in the Chemical Safety Report, given that the comparison of the derived DNEL with the results of the exposure assessment indicate that exposures are always well below the derived DNEL.

 

Although, Annex XI, Section 3 (June 1, 2015), states that “a DNEL derived from a screening test for reproductive/developmental toxicity shall not be considered appropriate to omit a prenatal developmental toxicity or a two-generation reproductive toxicity study”, paragraph 5 to Annexes IX and X states that “When, for certain endpoints, information is not provided for other reasons than those mentioned in Column 2 of this Annex or in Annex XI, this fact and the reasons shall also be clearly stated”

 

Therefore, as demonstrated in the CSR, the very low potential for exposure to the substance throughout its lifecycle due to its manufacture and use taking place in a rigorously contained system with emission minimisation controls, coupled with the risk characterisation ratios being significantly less than 1, are considered sufficient grounds to omit this study. Any potential extra information that would be gained from the performance of this test are outweighed by the opportunity to prevent unnecessary vertebrate animal testing.

 

The Registrant therefore proposes to waive the study required to address this endpoint.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

No adverse reproductive or developmental findings were observed up to the highest dose level tested during the study and therefore, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to reproductive and developmental toxicity.