Sodium long chain alkyl salicylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 October 2014 to 22 December 2014

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 10 weeks (males) or approximately 11 weeks (females)
- Weight at study initiation: (P) Males: 321 g; Females: 205 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: 26 November 2014 (males); 09, 12, 15, 17 and 22 December 2014 (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 5 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stirred during dosing of all animals. Formulations were heated to a maximum of 77.3 °C for approximately one hour to obtain visual homogeneity. Formulations were allowed to cool down to below 40 °C before dosing. Adjustment was made for specific gravity of the vehicle.

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: No data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- 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. During the lactation period, pups were kept with the dam until termination.

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 during the treatment phase 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 90 to 110 % 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) which was used for system control, data acquisition and data processing.

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 90 to 110 %.
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 90 to 110 % 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 0.9999.
No response at the retention time of the test material in the analytical blank samples was detected.
- Samples
Procedural recovery samples: The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. 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 90 and 110 %).
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 termination. Females were exposed for 41 to 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 15, 50 and 150 mg/kg/day
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 in which dosages of 30, 100 and 300 mg/kg/day were administered by daily oral gavage. No mortality occurred, and no toxicologically relevant clinical signs were noted. Stomach effects (red foci on the glandular stomach and/or irregular surface of the forestomach) were noted in two males at 100 mg/kg/day/day and in 3/5 males and 3/5 females at 300 mg/kg/day (also thickened duodenal wall was noted for one female at 300 mg/kg/day). Clinical biochemistry changes included higher alanine aminotransferase and alkaline phosphatase activities in both sexes, and higher glucose levels in males at 300 mg/kg/day. Males at this dose level additionally showed lower calcium and total protein levels. Males and females at 300 mg/kg/day showed higher liver weights (absolute and relative; relative weight approximately 15 % higher than controls). Body weight of males at 300 mg/kg/day was slightly reduced without treatment-related changes in food intake.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily animals were examined for mortality/viability. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.

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. 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 AND COMPOUND INTAKE:
- 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 AND COMPOUND INTAKE: 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. 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. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
-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 and all moribund animals 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. 62 and 67) were necropsied on post-coitum Day 25 (female with evidence of mating) or approximately 21 days after the last day of the mating period (female without evidence of mating). Animal no. 78 was euthanised in extremis.

GROSS NECROPSY
All animals were subjected to an external, thoracic and abdominal 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, testes, 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. The epididymides and testes were fixed in modified Davidson's solution and transferred to formalin after fixation for at least 24 hours.
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 Groups 1 and 4.
- Additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile or from which the female died before mating, to examine staging of spermatogenesis.
- The preserved organs and tissues of female no. 78 that was killed in extremis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of two Group 3 pairs (female no. 62 / male no. 22 - female not pregnant and female no. 67 and male no. 27 - not mated), and one Group 4 male (no. 38 - female no.78 sacrificed in extremis).
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. Pups found dead during the weekend were fixed in identified containers containing 70 % ethanol.

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, defects or cause of death were evaluated.

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:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

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)
One female (no. 78) treated at 150 mg/kg/day was sacrificed in extremis on Day 7 of the premating phase. Clinical signs noted on Days 6 and/or 7 included hunched posture, uncoordinated movements, abnormal gait, breathing difficulties, piloerection, chromodacryorrhoea and ptosis. Necropsy revealed an irregular surface of the forestomach (not supported by histopathological findings). No cause of morbidity could be established histopathologically.
No further mortality occurred during the study period.

CLINICAL SIGNS (PARENTAL ANIMALS)
No clinical signs of toxicity were noted during the observation period among animals surviving up to the scheduled necropsy.
Salivation seen after dosing among the dose groups in a dose-related manner was not considered 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 related to taste of the test material rather than a sign of systemic toxicity.
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 used 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 WEIGHTS (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.
Statistically significant lower food consumption after correction for body weight seen for females at 150 mg/kg/day between Days 11 and 14 post-coitum was considered to be unrelated to treatment since this change was not consistently seen over the treatment period.

MACROSCOPIC EXAMINATION (PARENTAL ANIMALS)
There were possibly test material-related gross observations in the stomach (forestomach, irregular surface) of 2 males at 50 mg/kg/day and 150 mg/kg/day and of the female that was sacrificed before the end of the study.
All other gross observations were considered to be incidental findings and not related to the test-material.
This includes the reduced size of testes and epididymides observed for animal 27 (microscopic correlate: massive tubular atrophy and massive reduced sperm).

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

MICROSCOPIC EXAMINATION (PARENTAL ANIMALS)
There were no test material-related microscopic observations, including in the stomach and liver. All 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.
Failure to sire or delivery of healthy pups of one pair at 50 mg/kg/day (male no. 27 / female no. 67) was related to a massive tubular atrophy (and absence of all spermatogenic stages) in the testes and massive reduced sperm in the epididymides. This correlated with a reduced size and lower weight of the testes and epididymides at necropsy. For the other pair at 50 mg/kg/day (male no. 22 / female no. 62) no cause could be found histopathologically for the failure to sire or delivery of healthy pups.
For one pair at 150 mg/kg/day (female no. 78 / male no. 38), failure to sire or delivery of healthy pups was due to sacrifice of the female on Day 7 of the premating phase. Since the incidence of failure to sire/ deliver healthy pups at these dose levels did not show a dose-related trend, this was considered to be unrelated to treatment.
The spermatogenic profiles of all other males examined were normal.

REPRODUCTION/DEVELOPMENTAL DATA (PARENTAL ANIMALS)
- Reproduction data
Mating, fertility and conception indices, pre-coital time, and number of corpora lutea and implantation sites were considered unaffected by treatment.
For female nos. 54, 55, 56 and 74, the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 5 or 6 of lactation.
- Developmental data
Gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were considered unaffected by treatment.
- Parturition/maternal care
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.

Dose descriptor:

NOAEL

Remarks:

parental, reproductive and developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes occurred in parental parameters up to the highest dose level tested.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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, and clinical signs, body weight and external macroscopy did not reveal any treatment-related findings.

MORTALITY (OFFSPRING)
Two pups of the control group, four pups at 50 mg/kg/day and two pups at 150 mg/kg/day were sacrificed in extremis, 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)
Incidental clinical symptoms of pups consisted of a wound above the right eye, moribund condition, and blue spots on the flanks. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHTS (OFFSPRING)
Body weights of pups were considered unaffected by treatment.

MACROSCOPY (OFFSPRING)
The nature and incidence of autolysis for one dead pup 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 (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

not specified

Table 1: Summary of Reproduction data

	Dose Group (mg/kg)
	0	15	50	150
Females paired	10	10	10	9
Females mated	10	10	9	9
Females pregnant	10	10	8	9
Females with living pups on Day 1	10	10	8	9
Mating index (%)	100	100	90	100
Fertility index (%)	100	100	80	100
Conception index (%)	100	100	89	100
Gestation index (%)	100	100	100	100

 

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	2	1	1	1
2	5	1	6	4
3	2	5	1	2
4	1	1	1	2
11	-	2	-	-
Median pre-coital time (days)	2	3	2	2
Mean pre-coital time (days)	2.2	4.4	2.2	2.6
N	10	10	9	9

 

Table 3: Summary of Corpora Lutea and Implantation Sites

Parameter		Dose Group (mg/kg)
		0	15	50	150
Corpora lutea	Mean SD N	13.5 4.0 10	13.9 1.5 10	13.5 2.0 8	13.6 4.9 9
Implantations	Mean SD N	11.5 3.3 10	11.9 2.1 10	12.1 2.9 8	10.9 3.5 9

SD = standard deviation

Conclusions:

Under the conditions of this study, a parental, reproduction and developmental No Observed Adverse Effect Level (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. The control group and the three treated groups each 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 41 to 54 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 (daily), body weight and food consumption (at least at weekly intervals), 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 formulation analysis showed that the formulations were prepared accurately and homogenously.

- Parental results

One female at 150 mg/kg/day was sacrificed in extremis on Day 7 of the premating phase. No cause of morbidity could be established histopathologically. This animal showed an irregular surface of the forestomach at necropsy which was not confirmed histopathologically. Given that no mortality or clinical signs of ill-health were noted for other animals of this dose group of either sex during any stage of the treatment phase, this death was considered to be unrelated to treatment with the test material.

Irregular surface of the forestomach was also noted for two males each at 50 mg/kg/day and 150 mg/kg/day. In the absence of correlating histopathological changes in the stomach or any supportive findings during the in-life phase (i.e. clinical signs, body weights and food intake), this necropsy finding was considered not adverse in nature.

Overall, it was considered that no adverse changes had occurred in parental parameters up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination of liver and stomach).

- Reproductive results

No reproduction 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, numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs).

- Developmental results

No developmental toxicity was observed up to the highest dose level tested (150 mg/kg/day). No treatment-related changes were noted in any of the 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, body weight and macroscopy).

Under the conditions of this study, a parental, reproduction and developmental No Observed Adverse Effect Level (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:

subchronic

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

In the key study, 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. The control group and the three treated groups each 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 41 to 54 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 (daily), body weight and food consumption (at least at weekly intervals), 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 formulation analysis showed that the formulations were prepared accurately and homogenously.

- Parental results

One female at 150 mg/kg/day was sacrificed in extremis on Day 7 of the premating phase. No cause of morbidity could be established histopathologically. This animal showed an irregular surface of the forestomach at necropsy which was not confirmed histopathologically. Given that no mortality or clinical signs of ill-health were noted for other animals of this dose group of either sex during any stage of the treatment phase, this death was considered to be unrelated to treatment with the test material.

Irregular surface of the forestomach was also noted for two males each at 50 mg/kg/day and 150 mg/kg/day. In the absence of correlating histopathological changes in the stomach or any supportive findings during the in-life phase (i.e. clinical signs, body weights and food intake), this necropsy finding was considered not adverse in nature.

Overall, it was considered that no adverse changes had occurred in parental parameters up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination of liver and stomach).

- Reproductive results

No reproduction 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, numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs).

- Developmental results

No developmental toxicity was observed up to the highest dose level tested (150 mg/kg/day). No treatment-related changes were noted in any of the 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, body weight and macroscopy).

Under the conditions of this study, a parental and reproduction No Observed Adverse Effect Level (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 under effective Strictly Controlled Conditions, 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 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 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:

27 October 2014 to 22 December 2014

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 weeks (females)
- Weight at study initiation: (P) 205 g (females)
- 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 last 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 09, 12, 15, 17 and 22 December 2014 (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 5 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stirred during dosing of all animals. Formulations were heated to a maximum of 77.3 °C for approximately one hour to obtain visual homogeneity. Formulations were allowed to cool down to below 40 °C before dosing. Adjustment was made for specific gravity of the vehicle.

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 during the treatment phase 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 90 to 110 % 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 Group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment Groups 2 and 4 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) which was used for system control, data acquisition and data processing.

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 90 to 110 %.
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 90 to 110 % 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 0.9999.
No response at the retention time of the test material in the analytical blank samples was detected.
- Samples
Procedural recovery samples: The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the Group 1 formulations, no test material was detected. The concentrations analysed in the formulations of Group 2, 3 and Group 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10 %).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: No data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- 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. During the lactation period, pups were kept with the dam until termination.

Duration of treatment / exposure:

Females were exposed for 41 to 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

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 in which dosages of 30, 100 and 300 mg/kg/day were administered by daily oral gavage. No mortality occurred, and no toxicologically relevant clinical signs were noted. Stomach effects (red foci on the glandular stomach and/or irregular surface of the forestomach) were noted in two males at 100 mg/kg/day/day and in 3/5 males and 3/5 females at 300 mg/kg/day (also thickened duodenal wall was noted for one female at 300 mg/kg/day). Clinical biochemistry changes included higher alanine aminotransferase and alkaline phosphatase activities in both sexes, and higher glucose levels in males at 300 mg/kg/day. Males at this dose level additionally showed lower calcium and total protein levels. Males and females at 300 mg/kg/day showed higher liver weights (absolute and relative; relative weight approximately 15 % higher than controls). Body weight of males at 300 mg/kg/day was slightly reduced without treatment-related changes in food intake.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily animals were examined for mortality/viability. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.

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. 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 AND COMPOUND INTAKE:
- 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 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: 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. 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 and all moribund animals were deeply anaesthetised using isoflurane and subsequently exsanguinated.
Females which delivered were necropsied on lactation Days 5-7. Females which failed to deliver (nos. 62 and 67) were necropsied on post-coitum Day 25 (female with evidence of mating) or approximately 21 days after the last day of the mating period (female without evidence of mating). Animal no. 78 was euthanised in extremis.

GROSS NECROPSY
All animals were subjected to an external, thoracic and abdominal 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, 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 Groups 1 and 4.
- The preserved organs and tissues of female no. 78 that was killed in extremis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, coagulation gland, ovaries, preputial gland, uterus, and vagina) of two Group 3 females (no. 62, not pregnant and female no. 67, not mated), and no.78 ,sacrificed in extremis.
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. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
-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 5-7 of lactation. Pups found dead during the weekend were fixed in identified containers containing 70 % ethanol.

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, defects or cause of death were evaluated.

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 group.
- 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:no effects

Details on maternal toxic effects:
MORTALITY (PARENTAL ANIMALS)
One female (no. 78) treated at 150 mg/kg/day was sacrificed in extremis on Day 7 of the premating phase. Clinical signs noted on Days 6 and/or 7 included hunched posture, uncoordinated movements, abnormal gait, breathing difficulties, piloerection, chromodacryorrhoea and ptosis. Necropsy revealed an irregular surface of the forestomach (not supported by histopathological findings). No cause of morbidity could be established histopathologically.
No further mortality occurred during the study period.

CLINICAL SIGNS (PARENTAL ANIMALS)
No clinical signs of toxicity were noted during the observation period among animals surviving up to the scheduled necropsy.
Salivation seen after dosing among the dose groups in a dose-related manner was not considered 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 related to taste of the test material rather than a sign of systemic toxicity.
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 used 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 WEIGHTS (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.
Statistically significant lower food consumption after correction for body weight seen for females at 150 mg/kg/day between Days 11 and 14 post-coitum was considered to be unrelated to treatment since this change was not consistently seen over the treatment period.

MACROSCOPIC EXAMINATION (PARENTAL ANIMALS)
There were possibly test material-related gross observations in the stomach (forestomach, irregular surface) of the female that was sacrificed before the end of the study.
All other gross observations were considered to be incidental findings and not related to the test-material.

MICROSCOPIC EXAMINATION (PARENTAL ANIMALS)
There were no test material-related microscopic observations, including in the stomach and liver. All 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.
Failure to sire or delivery of healthy pups of one pair at 50 mg/kg/day (male no. 27 / female no. 67) was related to a massive tubular atrophy (and absence of all spermatogenic stages) in the testes and massive reduced sperm in the epididymides. This correlated with a reduced size and lower weight of the testes and epididymides at necropsy. For the other pair at 50 mg/kg/day (male no. 22 / female no. 62) no cause could be found histopathologically for the failure to sire or delivery of healthy pups.
For one pair at 150 mg/kg/day (female no. 78 / male no. 38), failure to sire or delivery of healthy pups was due to sacrifice of the female on Day 7 of the premating phase. Since the incidence of failure to sire/ deliver healthy pups at these dose levels did not show a dose-related trend, this was considered to be unrelated to treatment.

REPRODUCTION/DEVELOPMENTAL DATA (PARENTAL ANIMALS)
- Reproduction data
Mating, fertility and conception indices, pre-coital time, and number of corpora lutea and implantation sites were considered unaffected by treatment.
For female nos. 54, 55, 56 and 74, the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 5 or 6 of lactation.
- Developmental data
Gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were considered unaffected by treatment.
- Parturition/maternal care
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.

Dose descriptor:

NOAEL

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:
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, and clinical signs, body weight and external macroscopy did not reveal any treatment-related findings.

MORTALITY (OFFSPRING)
Two pups of the control group, four pups at 50 mg/kg/day and two pups at 150 mg/kg/day were sacrificed in extremis, 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)
Incidental clinical symptoms of pups consisted of a wound above the right eye, moribund condition, and blue spots on the flanks. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHTS (OFFSPRING)
Body weights of pups were considered unaffected by treatment.

MACROSCOPY (OFFSPRING)
The nature and incidence of autolysis for one dead pup 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.

Basis for effect level:

other: No effects on any of the above parameters

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Summary of Developmental data

Parameter	Dose Group (mg/kg)
	0	15	50	150
Total number of litters	10	10	8	9
Duration of gestation (days) Mean SD N	21.8 0.6 10	21.6 0.5 10	21.5 0.8 8	21.4 0.5 9
Dead pups at first litter check Number of litters affected Total Mean SD N	1 1 0.1 0.3 10	0 0 0.0 0.0 10	0 0 0.0 0.0 8	0 0 0.0 0.0 9
Living pups at first litter check Percentage of males / females Total Mean SD N	45 / 55 107 10.7 3.4 10	47 / 53 109 10.9 3.2 10	48 / 52 97 12.1 3.4 8	49 / 51 96 10.7 3.6 9
Postnatal loss Percentage of living pups Number of litters affected Total Mean SD N	0.9 1 1 0.1 0.3 10	0.0 0 0 0.0 0.0 10	4.1 4 4 0.5 0.5 8	2.1 2 2 0.2 0.4 9
Viability index	99.1	100.0	95.9	97.9

SD = Standard deviation

 

Table 2: Summary of Pup Body Weights (g)

Lactation Day	Sex		Dose Group (mg/kg)
			0	15	50	150
1	M	Mean SD N	6.7 0.6 10	6.4 0.7 9	6.6 0.8 8	6.6 1.1 9
	F	Mean SD N	6.3 0.5 9	6.0 0.8 10	6.1 0.5 8	6.3 1.1 9
	M + F	Mean SD N	6.6 0.6 10	6.2 0.7 10	6.4 0.7 8	6.5 1.1 9
4	M	Mean SD N	9.8 0.7 10	8.9 1.1 9	9.8 1.3 8	9.4 1.7 9
	F	Mean SD N	9.3 0.6 9	8.4 1.4 10	8.9 1.2 8	9.0 2.0 9
	M + F	Mean SD N	9.7 0.8 10	8.7 1.2 10	9.4 1.2 8	9.2 1.8 9

SD = Standard deviation

Conclusions:

Under the conditions of this study, a parental, reproduction and developmental No Observed Adverse Effect Level (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. The control group and the three treated groups each consisted of 10 males and 10 females. Females were exposed for 41 to 54 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 (daily), body weight and food consumption (at least at weekly intervals), 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 formulation analysis showed that the formulations were prepared accurately and homogenously.

- Parental results

One female at 150 mg/kg/day was sacrificed in extremis on Day 7 of the premating phase. No cause of morbidity could be established histopathologically. This animal showed an irregular surface of the forestomach at necropsy which was not confirmed histopathologically. Given that no mortality or clinical signs of ill-health were noted for other animals of this dose group of either sex during any stage of the treatment phase, this death was considered to be unrelated to treatment with the test material.

Overall, it was considered that no adverse changes had occurred in parental parameters up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination of liver and stomach).

- Reproductive results

No reproduction 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, numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs).

- Developmental results

No developmental toxicity was observed up to the highest dose level tested (150 mg/kg/day). No treatment-related changes were noted in any of the 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, body weight and macroscopy).

Under the conditions of this study, a parental, reproduction and developmental No Observed Adverse Effect Level (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:

subchronic

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

In the key study, 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. The control group and the three treated groups each consisted of 10 males and 10 females. Females were exposed for 41 to 54 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 (daily), body weight and food consumption (at least at weekly intervals), 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 formulation analysis showed that the formulations were prepared accurately and homogenously.

- Parental results

One female at 150 mg/kg/day was sacrificed in extremis on Day 7 of the premating phase. No cause of morbidity could be established histopathologically. This animal showed an irregular surface of the forestomach at necropsy which was not confirmed histopathologically. Given that no mortality or clinical signs of ill-health were noted for other animals of this dose group of either sex during any stage of the treatment phase, this death was considered to be unrelated to treatment with the test material.

Overall, it was considered that no adverse changes had occurred in parental parameters up to the highest dose level tested (150 mg/kg/day).

No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination of liver and stomach).

- Reproductive results

No reproduction 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, numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs).

- Developmental results

No developmental toxicity was observed up to the highest dose level tested (150 mg/kg/day). No treatment-related changes were noted in any of the 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, body weight and macroscopy).

Under the conditions of this study, a parental and developmental No Observed Adverse Effect Level (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 under effective Strictly Controlled Conditions, 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.