Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Waiving (see Discussion).

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Regulation (EC) No. 1907/2006, Annex X, 8.7.3 Column 1, states that an extended one-generation reproduction toxicity study (EOGRTS, OECD 443, standard configuration) is required, using the most appropriate route of administration, and having regard to the likely route of human exposure. Regulation (EC) No. 1907/2006, Annex XI, states: ‘a registrant may adapt the standard testing regime in accordance with the general rules set out in Section 1 of this Annex’; where Section 1 addresses circumstances under which ‘testing does not appear scientifically necessary’. Annex XI, Section 1.2 covers the ‘weight of evidence’ principle that may be applied using ‘several independent sources of information’ to justify adapting the standard testing regime.  Therefore, in accordance with Annex XI, Section 1.2, the registrant has considered the need to perform an extended one-generation reproduction toxicity study. The summary below explains the rationale for the registrant’s conclusion that additional testing is not scientifically justified based on a review of the results of studies that cover reproductive, fertility and developmental parameters, and of other relevant information. In the 90-day Repeated Dose Toxicity Studies performed in two different species, rats and dogs (Hazleton 564/164; Hazleton 564/162) according to OECD 408 and 409, respectively, ovaries, testes with epididymides, uterus with vagina and cervix, mammary gland and prostate were examined macro-and microscopically. No relevant or treatment-related changes on reproductive organs were found.  In the pre-natal developmental toxicity study performed in the rat according to OECD 414 (Whitlow, 2014) a statistically significant increase in post-implantation loss (30.2% of implantation sites compared to 2.6% in the control group) was recorded in the high-dose group (90 mg/kg bw/day), which resulted in a significant decrease in the mean number of fetuses per dam in comparison to control animals. In the low- (10 mg/kg bw/day) and mid-dose group (30 mg/kg bw/day) post-implantation loss and the mean number of fetuses per litter were not affected by treatment with the test item. Macroscopic examination of the females did not reveal any abnormalities in any of the dose groups. The LOAEL for maternal toxicity was set at 90 mg/kg bw/day due to the apparent decreases in both the food consumption and body weight gain and the accompanied increase in post-implantation loss with an associated decrease in the number of fetuses per litter. No test-substance related maternal effects were noted in the low- and mid-dose group. Thus, a NOAEL for maternal toxicity of 30 mg/kg bw/day was derived. Skeletal examination of the fetuses on this study revealed abnormalities of the cervical vertebrae in the mid- and high-dose group. Therefore, the LOAEL for developmental toxicity was 30 mg/kg bw/day and the NOAEL was set at 10 mg/kg bw/day. In addition, the dose-dependently increased incidence of an altered texture of the cut surface of the eye lens, which is normally considered to be a process artifact, was especially high in the mid- and high-dose group and it could not be excluded that this was a possible effect of the test item. As the relevance of the effects observed for man is unclear the conduct of another prenatal developmental toxicity study in a second species is proposed in order to find out if the effects observed in rats are species-specific findings. For the time being, the conclusion with regards to classification and labelling for toxicity to reproduction (developmental toxicity) will be CLP: Category 2, H361. In conclusion, information is available from studies performed under several guidelines relevant to reproduction and fertility effects. There is no indication for reproduction and fertility effects. Therefore, referring to Regulation (EC) No. 1907/2006, Annex XI, Section 1.2, and for animal welfare reasons, performing an extended one-generation reproduction toxicity study (standard configuration or with additional modules) is not scientifically necessary and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified. Regulation (EC) No. 1907/2006, Annex X, 8.7.3 Column 1, states that an extended one-generation reproduction toxicity study (EOGRTS, OECD 443, standard configuration) is required, using the most appropriate route of administration, and having regard to the likely route of human exposure. Regulation (EC) No. 1907/2006, Annex XI, states: ‘a registrant may adapt the standard testing regime in accordance with the general rules set out in Section 1 of this Annex’; where Section 1 addresses circumstances under which ‘testing does not appear scientifically necessary’. Annex XI, Section 1.2 covers the ‘weight of evidence’ principle that may be applied using ‘several independent sources of information’ to justify adapting the standard testing regime.  Therefore, in accordance with Annex XI, Section 1.2, the registrant has considered the need to perform an extended one-generation reproduction toxicity study. The summary below explains the rationale for the registrant’s conclusion that additional testing is not scientifically justified based on a review of the results of studies that cover reproductive, fertility and developmental parameters, and of other relevant information. In the 90-day Repeated Dose Toxicity Studies performed in two different species, rats and dogs (Hazleton 564/164; Hazleton 564/162) according to OECD 408 and 409, respectively, ovaries, testes with epididymides, uterus with vagina and cervix, mammary gland and prostate were examined macro-and microscopically. No relevant or treatment-related changes on reproductive organs were found.  In the pre-natal developmental toxicity study performed in the rat according to OECD 414 (Whitlow, 2014) a statistically significant increase in post-implantation loss (30.2% of implantation sites compared to 2.6% in the control group) was recorded in the high-dose group (90 mg/kg bw/day), which resulted in a significant decrease in the mean number of fetuses per dam in comparison to control animals. In the low- (10 mg/kg bw/day) and mid-dose group (30 mg/kg bw/day) post-implantation loss and the mean number of fetuses per litter were not affected by treatment with the test item. Macroscopic examination of the females did not reveal any abnormalities in any of the dose groups. The LOAEL for maternal toxicity was set at 90 mg/kg bw/day due to the apparent decreases in both the food consumption and body weight gain and the accompanied increase in post-implantation loss with an associated decrease in the number of fetuses per litter. No test-substance related maternal effects were noted in the low- and mid-dose group. Thus, a NOAEL for maternal toxicity of 30 mg/kg bw/day was derived. Skeletal examination of the fetuses on this study revealed abnormalities of the cervical vertebrae in the mid- and high-dose group. Therefore, the LOAEL for developmental toxicity was 30 mg/kg bw/day and the NOAEL was set at 10 mg/kg bw/day. In addition, the dose-dependently increased incidence of an altered texture of the cut surface of the eye lens, which is normally considered to be a process artifact, was especially high in the mid- and high-dose group and it could not be excluded that this was a possible effect of the test item. As the relevance of the effects observed for man is unclear the conduct of another prenatal developmental toxicity study in a second species is proposed in order to find out if the effects observed in rats are species-specific findings. For the time being, the conclusion with regards to classification and labelling for toxicity to reproduction (developmental toxicity) will be CLP: Category 2, H361. In conclusion, information is available from studies performed under several guidelines relevant to reproduction and fertility effects. There is no indication for reproduction and fertility effects. Therefore, referring to Regulation (EC) No. 1907/2006, Annex XI, Section 1.2, and for animal welfare reasons, performing an extended one-generation reproduction toxicity study (standard configuration or with additional modules) is not scientifically necessary and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified.

Effects on developmental toxicity

Description of key information
Prenatal developmental toxicity study (OECD 414), rat: NOAELmaternal = 30 mg/kg bw/day, NOAELdevelopmental = 10 mg/kg bw/day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Apr - 14 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted Jan 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
dated Apr 2004
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
dated Aug 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHan™: WIST(SPF)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Horst, Netherlands
- Age (Day 0 post coitum): 12 weeks
- Weight (Day 0 post coitum): 215 - 262 g
- Housing: individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment
- Diet: pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: community tap-water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10- 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Apr 2013 To: 14 May 2013
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG 300)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily. The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle PEG 300 was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Concentration in vehicle: 2, 6 and 18 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: BCBG8285V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of each test item concentration were taken from the middle to confirm the stability (4 hours and 8 days at room temperature (20 ± 5 °C). Towards the end of the study, samples were taken from the middle to confirm concentration.The samples were analysed by HPLC with light-scattering detector (ELSD). Injected samples were quantified by comparing peak areas (sum of four peaks) of the test item with reference to the calibration curve.
The analysis of dose formulations revealed, that the test substance concentrations in the dose formulations ranged from 103.3% to 110.3% with reference to the nominal and were within the acceptance range of ±20% of the expected concentration, in all samples.
Homogenous distribution was confirmed because single results found did not deviate more than 1.1% from the corresponding mean values and met the specified acceptance criterion of ≤ 15%. Therefore, a correct dose formulation preparation was confirmed. The test item was found to be stable in application formulations when kept for 8 days at room temperature (20 ± 5 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: copulation plug or sperm in vaginal smear referred to as day 0 post coitum
Duration of treatment / exposure:
Day 6 - Day 20 post coitum (p.c.)
Frequency of treatment:
daily, 7 days/week
Duration of test:
15 days of gestation
Remarks:
Doses / Concentrations:
10, 30 and 90 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on the results of a foregoing dose range-finding prenatal developmental toxicity study (experimental period: 06 Dec 2012 – 04 Jan 2013) in the same testing labor under very similar testing conditions as in the main study in regard to husbandry, dose formulation, study schedule, mating and observations. 5 mated Han Wistar rats per group were administered the test substance via gavage at concentrations of 100, 300 and 1000 mg/kg bw/day from day 6 – 20 post coitum. A control group received the vehicle corn oil alone.
In the high- and mid-dose group 5/5 and 2/5 dams, respectively were found dead or were killed for ethical reasons. At necropsy 2 animals of the high-dose group had fluid in the colon. No abnormal findings were observed at necropsy in the other animals, which died before scheduled necropsy. Clinical signs among animals surviving until Caesarean section included salivation, secretion (fluid) in the anal region, bedding in the mouth and ruffled fur. In the low-dose group food consumption was statistically significantly reduced after treatment start over days 6-9 and 9-12 p.c. and remained reduced without statistical significance for the rest of the treatment period. Body weight and body weight gain of the low-dose group dams were statistically significant reduced from day 11 p.c. onwards.
In the mid-dose group the 3 surviving animals had resorptions only. In the low-dose group four females were pregnant and one female had resorptions only. Post-implantation loss was statistically significant increased in the low-dose group, which resulted in a reduced number of fetuses per litter. No abnormal macroscopical findings were observed in the control group or in the low-dose group during maternal necropsy. External examination of the fetuses of the low-dose group did not reveal external abnormalities or variations. No test-item related effects on fetal sex ratios and on fetal body weights were observed. Since severe maternal toxicity was observed at doses of 300 and 1000 mg/kg bw/day, dose levels of up to 90 mg/kg bw/day were considered appropriate for a main prenatal developmental toxicity study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was recorded twice daily. Cage-side clinical observations were performed once daily during acclimatization and up to day of necropsy.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 21 post coitum (p.c.)

FOOD CONSUMPTION: Yes
- Food consumption was recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 abd 18-21 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all internal organs with emphasis on the uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (detailed examination of major blood vessels, the head, the heart and the kidneys)
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
- mean and standard deviations of various data were calculated
- Dunnett-test (many to one t-test) based on a pooled variance estimate: applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex
- Steel-test (many-one rank test): applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution
- Fisher's exact-test: applied if the variables could be dichotomized without loss of information
Historical control data:
Historical control data were provided to allow comparison with concurrent controls. The historical control data comprises data of 6 embryofetal development studies performed at the testing laboratory throughout the year 2010. Detailed data on rat (HanRcc: WIST) reproduction, visceral examination (microdissection), skeletal examination (bone and cartilage findings) were included.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: high dose: post-implantation loss, decrease in body weight/gain, decrease in food consumption

Details on maternal toxic effects:
PERFORMANCE OF MATED FEMALES
Two animals each (2/22) of the medium and high dose group were not pregnant. In the control group one female had resorptions only. 21, 22, 20 and 20 females in the control, low, medium and high dose group, respectively had live fetuses at termination (table 1). Only dams with at least one live fetus at caesarean section were used for the calculation of food consumption, body weight gain and corrected body weight gain data.

MORTALITY
All females survived until the scheduled necropsy on gestation day 21.

CLINICAL SIGNS
No clinical symptoms or signs were observed at any dose level during the course of the study, that were considered to be related to toxicity of the test item. One female in the high dose group had breathing noises for two days (GD 8 and 9) during the treatment period. Since only one female was affected, this was considered to be incidental.

BODY WEIGHT
In the 10 and 30 mg/kg bw/day dose group, no test item-related effects were observed on body weight, body weigh gain or corrected body weight gain (corrected for the weight of the gravid uterus) in comparison with control group.
In the 90 mg/kg bw/day dose group, mean body weight gain was statistically significantly reduced on days 4 and 7 to 8 p.c. and from day 10 p.c. until the end of the study (table 1). Absolute body weight was statistically significantly decreased from day 19 p.c. onwards. Corrected body weight gain was reduced without statistical significance (mean +6.6% compared to +10.7% in the control group). These reductions were considered to be test item-related.

FOOD CONSUMPTION
The amount of food consumed by females in the high dose group decreased following the start of dosing, when compared to pre-dose values and control animals, until necropsy on day 21. This reduction in food consumption achieved statistical significance from days 12 - 15 p.c. until the end of the study (table 1). Over the treatment period (days 6 - 21 p.c.), mean food consumption was -9.4% for the high dose group when compared to the control group (mean over days 6-21: control: 23.3 g/animal/day; high dose group: 21.1 g/animal/day). There was no effect of treatment on the amount of food consumed by females in the low and medium dose group over the course of the study.

REPRODUCTION DATA (table 2)
No changes were noted within the number of copora lutea between treatment and control groups.
In the high dose group, a statistically significant increase in post-implantation loss (30.2% of implantation sites compared to 2.6% in the control group) was recorded. This resulted in a statistically significant decrease in the mean number of fetuses per dam, when compared to control animals (high dose: 9.3 fetuses per dam; control: 12.6 fetuses per dam). This was considered to be a test item-related effect.
In the low and medium dose group, post-implantation loss (low dose: 1.3% of implantation sites; medium dose: 3.9% of implantation sites) and the mean number of fetuses per litter (low dose: 13.6; medium dose: 12.5) were not affected by treatment with the test item.
Although pre-implantation loss was statistically significantly increased in the medium and high dose group, resulting in a statistically significant decrease in the mean number of implantation sites, this was considered to be incidental since implantation is considered to have occurred prior to the start of dosing.

PATHOLOGY
Macroscopic examination revealed no findings in any female rat at any dose level.
Dose descriptor:
LOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: high dose: external abnormalities of head, decrease in fetus bodyweight, altered texture of cut surface of eye lens, cervical vertebra and cranial bone abnormalities; medium dose: altered texture of cut surface of eye lens, cervical vertebra abnormalities

Details on embryotoxic / teratogenic effects:
EXTERNAL ABNORMALITIES AND VARIATIONS
There was a higher incidence of fetuses with external abnormalities seen in the high dose group, with 5 fetuses in 4 litters affected (table 2). This may be an indication of a slight disturbance in the development of the fetuses, as all five fetuses were affected with abnormalities of the head including slightly misshapen head, no skin over head, missing eyes and nasal opening, cleft lip and clear membrane over part of head. Although all fetuses were not affected by the same abnormality, a possible association to treatment could not be ruled out.
No abnormal findings were recorded for the control and medium dose group. In the low dose group a shortened lower jaw and a closed mouth was recorded.

SEX RATIOS
No test item-related effects on the sex ratio of the fetuses were noted in any group.
The proportion of male fetuses was 54.9%, 47.0%, 52.2% and 50.3% in order of ascending dose level (table 2).

BODY WEIGHTS
In the medium and high dose groups, the mean body weights calculated on an individual basis of the male and female fetuses combined (control: 4.9 ± 0.4 g; low dose: 4.8 ± 0.4 g; medium: 4.7 ± 0.5 g, high dose: 4.7 ± 0.4 g) as well as for the male fetuses in the high dose group (control: 5.0 ± 0.4 g; high dose: 4.8 ± 0.4 g), were slightly but statistically significantly reduced (table 2). Although the mean weights were within the range of the historical control data, since the mean number of fetuses per litter in the high dose group was reduced, it would be expected that these fetuses would be heavier. Therefore this slight reduction in body weight in the high dose group compared to the control group was considered to be a slight effect of the test item. No test item-related effects were observed in the fetuses in the low dose group.

VISCERAL ABNORMALITIES AND VARIATIONS
During visceral examination of the fetuses, findings were noted in 47, 34, 53 and 62% of examined fetuses in the control, low, medium and high dose group, respectively.
Malformations including severe bilateral dilatation of the lateral ventricle of the brain with a small eye that was severely reduced in size was seen in 1 fetus in the group given 90 mg/kg bw/day (table 3). However, as one pup in the control group was also found to have severe bilateral dilatation of the lateral ventricle of the brain, it was considered that these abnormalities were not associated with maternal administration of the test item. One incidence of anal atresia was seen in one fetus in the group given 90 mg/kg bw/day, and situs inversus was recorded for one fetus in the group given 10 mg/kg bw/day. However, as both instances were isolated findings, these were not considered to be test item-related.
The incidence of a malpositioned testis (classified as a variation) was slightly outside the range of the historical control data at 90 mg/kg bw/day and it could not be excluded that this was a test item-related effect (1, 4, 2 and 6 incidences seen in 1, 3, 2 and 6 litters at 0, 10, 30 or 90 mg/kg bw/day).
It should be noted that an altered texture of the cut surface of the eye lens is normally considered to be a process artifact. However, the incidence in all groups receiving the test item was dose-dependently increased (control: 0%, low dose: 6%, medium dose: 23%, high dose: 60% of fetuses examined). This incidence, in particular at 30 and 90 mg/kg bw/day was especially high and it could not be excluded that this was a possible effect of the test item (table 3).
No further test item-related findings were observed from the visceral examinations.

BONE CARTILAGE ABNORMALITIES AND VARIATIONS
During skeletal examination of the fetuses, findings were noted in 18, 10, 26 and 36% of examined fetuses in the control, low, medium and high dose group, respectively. Abnormalities were observed in 1 fetus from 1 litter, 2 fetuses from 2 litters, 3 fetuses from 3 litters and in 8 fetuses from 8 litters at 0, 10, 30 or 90 mg/kg bw/day, respectively.
A dose related increase in the incidence of cervical vertebra abnormalities was apparent, with 0, 1, 3 and 7 incidences seen in 0, 1, 3 and 7 litters at 0, 10, 30 or 90 mg/kg bw/day, respectively (table 4). The high incidence of abnormalities seen in the groups given 30 or 90 mg/kg bw/day, spread throughout the litters, were considered to be a test item-related effect. The single incidence of split ventral arch in cervical vertebra 1 seen in the group given 10 mg/kg bw/day, can be considered to be isolated and incidental, since this fetus had a low body weight and this finding may occur spontaneously. Incidences of cranial bone abnormalities (including absent, fused and/or misshapen, palate cleft or misshapen, hyoid arch structure absent or duplicated) were seen 1 fetus from 1 litter in each of the groups given 0, 10 or 30 mg/kg/day and in 5 fetuses from 5 litters at 90 mg/kg bw/day (table 4). Due to the increased number of incidences seen in the high dose and the dispersal of the affected fetuses across litters, it is considered that this increase in the number of abnormalities seen is attributable to administration of the test item at 90 mg/kg bw/day. At 0 and 30 mg/kg/day, the affected fetus was found to have a duplicated greater horn hyoid arch. This finding was considered to be incidental. At 10 mg/kg/day, the affected fetus had skull findings similar to those at 90 mg/kg/day. However, since this finding was not observed in any fetus in group 3 and the affected fetus did not have any cervical vertebral abnormalities present in the group 3 and 4 fetuses, it was considered to be incidental.
The remaining abnormalities observed occurred in isolated fetuses and were therefore considered to be incidental. Variations of the ribs (cervical rib and wavy rib) as well as fused costal cartilages were outside the range of the historical control data in the high dose group and were therefore considered to be test item-related.
All remaining variations were within the range of the historical control data and/or were not observed in a dose-dependent pattern.

OSSIFICATION AND SUPERNUMERARY RIBS
There was a statistically significant increase in the incidence of incomplete ossification in the cranium as well as supernumerary rudimentary ribs in the high dose group on both a litter and a fetus basis (table 4). These increased incidences were considered to be test item-related.
No further effects of the test item were observed.

ADDITIONAL CARTILAGE VARIATIONS
There was a statistically significant increase in the incidence of a long ventral plate at 90 mg/kg bw/day on both a litter and a fetus basis. The incidence was outside the range of the historical control data and was therefore considered to be a test item-related effect.
No further findings were observed that were considered to be test item-related.
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Maternal effects

 

Historical control (2010)

0 mg/kg bw/day

10 mg/kg bw/day

30 mg/kg bw/day

90 mg/kg bw/day

Performance of Mated Females

No. of mated females

20-24

22

22

22

22

Not pregnant

0-1

0

0

2

2

Resorptions only

--

1

0

0

0

Number of females with live fetuses at termination

--

21

22

20

20

Food Consumption (g/animal/day)

Days 0-3

 

21.9 ± 2.3

21.1 ± 1.8

21.7± 2.2

21.7± 2.2

Days 3-6

 

21.7 ± 2.6

21.4 ± 2.3

21.4 ± 1.7

21.9 ± 1.3

Days 6-9

 

21.2 ± 2.8

21.0 ± 2.2

20.5 ± 2.0

19.7 ± 2.1

Days 9-12

 

22.4±2.6

22.0± 2.0

21.4± 2.1

20.8± 2.0

Days 12-15

 

23.3 ± 2.3

23.1 ± 2.5

23.0 ± 1.9

20.9 ± 3.2**

Days 15-18

 

25.1± 2.9

24.5± 2.3

24.0 ± 2.4

22.7± 1.9**

Days 18-21

 

24.5± 3.2

23.5±3.3

23.6± 3.1

21.5± 4.5*

Mean of mean over gestation period

 

22.9

22.3

22.2

21.4

Body weight gain (%)

Day 0

 

-7 ± 2

-6 ± 2

7 ± 2

8 ± 2

Day 1

 

-6 ± 1

-5 ± 12

-6 ± 1

-6 ± 1

Day 2

 

-4 ± 1

-3 ± 2

-4 ± 1

-4 ± 1

Day 3

 

-3 ± 1

-3 ± 1

-3 ± 1

-3 ± 1

Day 4

 

-2 ± 1

-2 ± 1

-2 ± 1

-3 ± 1*

Day 5

 

-1 ± 1

-1 ± 1

-1 ± 1

-1 ± 1

Day 6

 

0 ± 0

0 ± 0

0 ± 0

0 ± 0

Day 7

 

2 ± 1

1 ± 1

1 ± 1

0 ± 2**

Day 8

 

3 ± 1

2 ± 1

2 ± 1

1 ± 2**

Day 9

 

3 ± 1

3 ± 1

3 ± 1

2 ± 2

Day 10

 

5 ± 1

6 ± 1

5 ± 2

3 ± 2

Day 11

 

7 ± 1

8 ± 2

7 ± 1

6 ± 2**

Day 12

 

10 ± 2

10 ± 2

9 ± 2

7 ± 2**

Day 13

 

11 ± 2

11 ± 2

11 ± 2

8 ± 3**

Day 14

 

13 ± 2

13 ± 2

13 ± 2

9 ± 4**

Day 15

 

16 ± 3

17 ± 2

15 ± 2

11 ± 4**

Day 16

 

19 ± 4

20 ± 3

18 ± 3

14 ± 4**

Day 17

 

24 ± 4

25 ± 3

23 ± 4

17 ± 6**

Day 18

 

29 ± 4

31 ± 4

28 ± 4

21 ± 6**

Day 19

 

34 ± 4

36 ± 4

33 ± 6

24 ± 8**

Day 20

 

39 ± 5

42 ± 5

38 ± 7

28 ± 9**

Day 21

 

44 ± 6

46 ± 5

42 ± 8

30 ± 11**

 

Table 2: Reproduction data

 

Historical control (2010)

0 mg/kg bw/day

10 mg/kg bw/day

30 mg/kg bw/day

90 mg/kg bw/day

Corpora lutea

Corpora lutea (total/No. of dams with live fetuses)

13.7 – 15.0

13.2 ± 2

14.2 ± 1.5

14.3 ± 1.9

14.1 ± 1.8

Pre-implantation loss

Pre-implantation loss, total (% of corpora lutea)

1.9 – 10.5

6 (2.2)

9 (2.9)

26 (9.1##)

17 (6.0#)

Implantation sites, total (% of corpora lutea)

262 – 319

(89.5 – 98.1)

271 (97.8)

304 (97.1)

259 (90.9##)

265 (94.0#)

Post-implantation loss

Post-implantation loss, total (% of implantation sites)

20 – 30

(6.8 – 11.4)

7 (2.6)

4 (1.3)

10 (3.9)

80 (3.2##)

Post-implantation loss, mean per dam

1.0 – 1.5

0.3 ± 0.6

0.2 ± 0.9

0.8 ± 0.8

4.0 ± 4.5+

embryonic/fetal deaths, total

20 - 30

7

4

10

80

embryonic/fetal deaths: embryonic resorptions, total (% of implantation sites)

20 – 29

(6.8 – 11.1)

6 (2.2)

4 (1.3)

8 (3.1)

79 (29.8##)

embryonic/fetal deaths: embryonic resorptions, mean per dam

1.0 – 1.5

0.3 ± 0.5

0.3 ± 0.9

0.4 ± 0.7

4.0 ± 4.5 (+)

Fetuses

Total fetuses (% of implantation sites)

233-295

(88.6 – 93.2)

264 (97.4)

300 (98.7)

249 (96.1)

185 (69.8##)

Total fetuses, mean per dam

11.1 13.0

12.6 ± 2.0

13.6 ± 1.5

12.5 ± 3.6

9.3 ± 4.0 (+)

Dead fetuses

0

0

0

0

0

Abnormal fetuses, total (% of fetuses)

0 – 1

(0 – 0.4)

0

1 (0.3)

0

5 (2.7%#) (4 dams affected)

Sex, % males

47.9 – 55.9

54.9

47.0#

52.2

50.3

Weights (g) of live fetuses, mean total (individual basis)

4.7 - 4.9

4.9 ± 0.4

4.8 ± 0.4

4.7 ± 0.5**

4.7 ± 0.4**

Weights (g) of live fetuses, mean males (individual basis)

4.8 – 4.9

5.0 ± 0.4

5.0 ± 0.4

4.9 ± 0.5

4.8 ± 0.4*

Weights (g) of live fetuses, mean females (individual basis)

4.6 – 4.8

4.7 ± 0.4

4.7 ± 0.3

4.6 ± 0.4

4.6 ± 0.4

*/**: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**)       

#/## : Fisher's Exact Test significant at level 5% (#) or 1% (##)

+ : Steel Test significant at level 5%

Table 3: Visceral examination of fetuses

 

Historical control (2010)

0 mg/kg bw/day

10 mg/kg bw/day

30 mg/kg bw/day

90 mg/kg bw/day

Visceral Abnormalities and Variations

Visceral abnormalities (N (%))

Eye small severe

0

0

0

0

Fetus: 1 (1)

Litter: 1 (5)

Brain ventricle dilated severe

0

Fetus: 1 (1)

Litter: 1 (5)

0

0

Fetus: 1 (1)

Litter: 1 (5)

Situs inversus

0

0

Fetus: 1 (1)

Litter: 1 (5)

0

0

Anal atresia

No data

0

0

0

Fetus: 1 (1)

Litter: 1 (5)

Visceral variations (N (%))1

Testis malpositioned

Fetus: 0-3 (0-2)

Litter: 0-2 (0-9)

Fetus: 1 (1)

Litter: 1 (5)

Fetus: 4 (3)

Litter: 3 (14)

Fetus: 2 (2)

Litter: 2 (10)

Fetus: 6 (6)

Litter: 6 (30)

Assumed / probable process artefacts (N (%))1

Eye lens cut surface altered texture

0

0

Fetus: 9 (6)

Litter: 6 (27)

Fetus: 31 (23)

Litter: 15 (75)

Fetus: 58 (60)

Litter: 19 (95)

1: only test-substance associated effects were listed

 

Table 4: Skeletal examination of fetuses

 

0 mg/kg bw/day

10 mg/kg bw/day

30 mg/kg bw/day

90 mg/kg bw/day

Bone and Cartilage Abnormalities

Bone and cartilage abnormalities (N (%))

Skull cranial bone absent, fused and/or misshapen, palate cleft or misshapen, hyoid arch structure absent or duplicated

Fetus: 1 (1)

Litter: 1 (5)

Fetus: 1 (1)

Litter: 1 (5)

Fetus: 1 (1)

Litter: 1 (5)

Fetus: 5 (6)

Litter: 5 (25)

Cervical vertebra ventral arch, body or dorsal arch absent, fused (to odontoid process or other vertebral structure), misshapen, interrupted, short and/or split

0

Fetus: 1 (1)

Litter: 1 (5)

Fetus: 3 (3)

Litter: 3 (15)

Fetus: 7 (8)

Litter: 7 (35)

Thoracic vertebra body and/or arch absent, fused,

misshapen and/or thick

0

Fetus: 1 (1)

Litter: 1 (5)

0

Fetus: 1 (1)

Litter: 1 (5)

Rib branched, misshapen and/or thick

0

Fetus: 1 (1)

Litter: 1 (5)

0

Fetus: 1 (1)

Litter: 1 (5)

Pectoral or forelimb bone bent or short

0

0

0

Fetus: 1 (1)

Litter: 1 (5)

Ossification and supernumerary ribs

Cranium, incompletely ossified (on a litter basis, N (%))

OS occipitale

1 (5%)

3 (14%)

2 (10%)

6 (30%)#

Os parietale, bilateral

2 (10%)

3 (14%)

6 (30%)

9 (45%)#

Os interparietale

1 (5%)

6 (27%)

4 (20%)

8 (40%)##

Os parietale, left

0

1 (5%)

0

0

Os hyoideum

0

2 (9%)

0

1 (5%)

Os frontale, left

2 (10%)

3 (14%)

4 (20%)

8 (40%)#

Os frontale, right

2 (10%)

3 (14%)

4 (20%)

8 (40%)#

Os nasale, left

1 (5%)

1 (5%)

1 (5%)

2 (10%)

Os nasale, right

1 (5%)

1 (5%)

1 (5%)

2 (10%)

Cranium, incompletely ossified (on a fetus basis, N (%))

Os occipitale

1 (1%)

3 (2%)

2 (2%)

6 (7%)#

Os parietale, bilateral

2 (2%)

4 (3%)

8 (7%)#

16 (18%)##

Os interparietale

1 (1%)

7 (5%)

5 (4%)

10 (11%)##

Os parietale, left

0

1 (1%)

0

0

Os hyoideum

0

2 (1%)

0

1 (1%)

Os frontale, left

2 (2%)

3 (2%)

5 (4%)

10 (11%)##

Os frontale, right

2 (2%)

3 (2%)

5 (4%)

10 (11%)##

Os nasale, left

1 (1%)

1 (1%)

1 (1%)

2 (2%)

Os nasale, right

1 (1%)

1 (1%)

1 (1%)

2 (2%)

Supernumerary ribs (on a litter basis, N (%))

Supernumerary, one rib(s), left

1 (5%)

2 (9%)

1 (5%)

2 (10%)

Supernumerary, one rudimentary rib(s), left

5 (24%)

8 (36%)

8 (40%)

13 (65%)##

Supernumerary, one rib(s), right

1 (5%)

2 (9%)

2 (10%)

1 (5%)

Supernumerary, one rudimentary rib(s), right

5 (24%)

9 (41%)

7 (35%)

11 (55%)#

Additional cartilage variation: cartilaginous cervical vertebrae (on a litter basis, N (%))

Shortened, ventral plate, left

0

1 (5%)

1 (5%)

0

Shortened, ventral plate, right

0

1 (5%)

1 (5%)

0

Long, ventral plate, left

0

2 (9%)

1 (5%)

5 (25%)#

Long, ventral plate, right

1 (5%)

1 (5%)

3 (15%)

6 (30%)#

Additional cartilage variation: cartilaginous cervical vertebrae (on a fetus basis, N (%))

Shortened, ventral plate, left

0

1 (1%)

1 (1%)

0

Shortened, ventral plate, right

0

1 (1%)

0

0

Long, ventral plate, left

0

2 (1%)

1 (1%)

6 (7%)##

Long, ventral plate, right

1 (1%)

1 (1%)

3 (3%)

8 (9%)##

#/## : Fisher's Exact Test significant at level 5% (#) or 1% (##)

 

Conclusions:
The test substance led to developmental effects in rats under the experimental conditions of the study. The relevance of the effects observed for man is unclear.
CLP: Category 2, H361d
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequateand reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The effects of Atmer 163 (CAS 97925-95-6) on the pregnant rat and development of the embryo and fetus was examined in a GLP-Guideline study according to OECD 414 (Harlan D61532). 22 mated females per group were treated by gavage with the test substance at doses of 10, 30 and 90 mg/kg bw/day from day 6 – 20 post coitum (p.c.). The dose selection was determined in a dose-range finding prenatal developmental toxicity study (Harlan D61521) A control group received the vehicle PEG 300 alone. All females were sacrificed on day 21 p.c. and the fetuses were removed by Caesarean section. No mortality occurred until the scheduled necropsy and no clinical signs were noted at any dose level during the study, which were considered to be test substance-related. Food consumption of the high-dose group was decreased following the start of dosing until necropsy on day 21, achieving statistical significance from period days 12-15 p.c. onwards. This decrease in food consumption was accompanied by a statistically significant decrease in mean body weight and body weight gain in the high-dose group during the dosing period. Corrected body weight gain (corrected for the weight of the gravid uterus) was reduced without statistical significance (mean +6.6% compared to +10.7% in control group). A statistically significant increase in post-implantation loss (30.2% of implantation sites compared to 2.6% in the control group) was recorded in the high-dose group, which resulted in a significant decrease in the mean number of fetuses per dam in comparison to control animals. In the low- and mid-dose group post-implantation loss and the mean number of fetuses per litter were not affected by treatment with the test item. Macroscopic examination of the females did not reveal any abnormalities in any of the dose groups.

Observations of the fetuses after Caesarean section revealed a higher incidence of fetuses with external abnormalities in the high-dose group with 5 fetuses in 4 litters affected. This may be an indication of a slight disturbance in the development of the fetuses, as all five fetuses were affected with abnormalities of the head including slightly misshapen head, no skin over the head, missing eyes and nasal opening, cleft lip and clear membrane over part of the head. No abnormal findings were recorded for the control and medium dose group. In the low dose group a shortened lower jaw and a closed mouth was observed. No test item-related effects on the sex ratio of the fetuses were noted in any dose group. Mean body weights of the high-dose group calculated on an individual basis of the male and female fetuses combined, as well as for the male fetuses alone, were slightly but statistically significantly reduced and considered as a test-item related effect. Although these mean weights were within the range of the historical control data, a higher fetus weight was expected due the reduced mean number of fetuses per litter in the high-dose group. No test item-related effects on body weight were observed in the fetuses in the low- and mid-dose group. During visceral examinations of the fetuses the incidence of a malpositioned testis (classified as a variation) was slightly outside the range of the historical control data in the high-dose group and it could not be excluded that this was a test-item-related effect. Moreover, an altered texture of the cut surface of the eye lens was noted and is normally considered to be a process artifact. However, the incidence in all groups receiving the test substance was dose- dependently increased. This incidence, in particular in the mid- and high-dose group was especially high and it could not be excluded that this was a possible effect of the test item. During skeletal examinations abnormalities were observed in 1 fetus from 1 litter, 2 fetuses from 2 litters, 3 fetuses from 3 litters and in 8 fetuses from 8 litters in order of ascending dose groups. There was an increased incidence of cervical vertebra abnormalities in the mid- and high-dose groups spread throughout the litters. Incidences of cranial bone abnormalities (including absent, fused and/or misshapen, palate cleft or misshapen, hyoid arch structure absent or duplicated) were increased in the high dose. Variations of the ribs (cervical rib and wavy rib) as well as fused costal cartilages were outside the range of the historical control data in the high dose group. All remaining findings were isolated or were within the range of the historical control data and/or were not observed in a dose-dependent pattern. In regard to ossification and supernumerary ribs, there was a statistically significant increase in the incidence of incomplete ossification in the cranium as well as supernumerary rudimentary ribs in the high dose group on both a litter and a fetus basis. These increased incidences were considered to be test item-related. Additional cartilage variations included a statistically significant increase in the incidence of a long ventral plate in the high-dose group on both a litter and a fetus basis. The incidence was outside the range of the historical control data and was therefore considered to be a test item-related effect.

In conclusion, the LOAEL for maternal toxicity was set at 90 mg/kg bw/day due to the apparent decreases in both the food consumption and body weight gain, the accompanied increase in post-implantation loss with an associated decrease in the number of fetuses per litter. No test-substance related maternal effects were noted in the low- and mid-dose group. Thus, a NOAEL for maternal toxicity of 30 mg/kg bw/day was derived.

Skeletal examination of the fetuses on this study revealed abnormalities of the cervical vertebrae in the mid- and high-dose group. Therefore, the LOAEL for developmental toxicity was 30 mg/kg bw/day and the NOAEL was set at 10 mg/kg bw/day. In addition, the dose-dependently increased incidence of an altered texture of the cut surface of the eye lens, which is normally considered to be a process artifact, was especially high in the mid- and high-dose group and it could not be excluded that this was a possible effect of the test item.

References:

Whitlow (2013). Atmer 163: Dose Range-Finding Developmental Toxicity Study in the Han Wistar Rat. Testing laboratory: Harlan Laboratories Ltd., Füllinsdorf, Switzerland. Report no.: D61521. Owner company: Croda Europe Ltd., UK. Report data: 2013-07-23

Justification for classification or non-classification

The test substance led to developmental effects in Han-Wistar rats at administered doses of 30 mg/kg bw/d and above under the experimental conditions of the study. The relevance of the effects observed for man is unclear.

The conduct of another prenatal developmental toxicity study in a second species is proposed in order to find out if the effects observed in rats are species-specific findings.

For the time being, the conclusion with regards to classification and labelling for toxicity to reproduction (developmental toxicity) will be

CLP: Category 2, H361d.