O-isopropyl ethylthiocarbamate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction: OECD 422; Crl:CD rats; NOAEL(P0): 31mg/kg bw/day; LOAEL(F1): ca. 31mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories,Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Males/females: Both 57 days
- Weight at study initiation: 163 - 209 g
- Fasting period before study: 16 hours
- Housing: kept individually in MAKROLON cages (type III plus)
- Diet (e.g. ad libitum): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany; served as food ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg b.w./day

Details on mating procedure:

- M/F ratio per cage: Sexually mature male and female rats were randomly paired for mating.1 male and 1 female animal were placed in one cage during the dark period.
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: Each morning the females were examined for the presence of sperm or a vaginal plug. If findings were negative, mating was repeated. The day of conception (day 0 of gestation) was considered to be the day on which sperm was found.
- Further matings after two unsuccessful attempts: In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered. This procedure was repeated until at least 8 pregnant dams were available for each group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples of approx. 2 x 1 mL were taken at the following time points and stored at ≤ minus 20°C until analysis at LPT:
Start of treatment period Concentration and stability Immediately after preparation of the test item vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature:
3 samples/dose level group (groups 2 to 4)
Number of samples: 3 x 3 = 9
Homogeneity
At start of administration, during (middle) administration and before administration to the last animal of each dose level group:
3 samples/dose level group (groups 2 to 4).
Number of samples: 3 x 3 = 9
End of treatment period Concentration
During treatment with the test item always before administration to the last animal/dose level
group: 1 sample/dose level group (groups 2 to 4).
Number of samples: TD 26 (1 x 3) = 3
Number of samples: TD 39 (1 x 3) = 3
Sum of all samples: 24
Sum of all aliquots: 48
The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.

Duration of treatment / exposure:

Males: The daily administration of the test item started two weeks before mating and lasted until the
day before sacrifice (considering a minimum total dosing period of at least 28 days).
Females: The daily administration of the test item started two weeks before mating and lasted up to at least day 3 of lactation.

Frequency of treatment:

once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 10-11 weeks

Dose / conc.:

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

Remarks:

The batch no. 0001026602 used in this study had a different concentration of the active substance Thionocarbamate than stated in the original certificate of analysis: GLP-Certificate of October 4th 2012 with 95.7% purity instead of 92.9% stated earlier (Certificate of June 6th 2012). Based on a purity of 92.9%, a correction factor of 1.08 was used in this study. Consequently, the actually administered dosages for the groups were slightly higher (31, 103 and 309 mg IPETC (DANAFLOATTM 262)/kg b.w./day) than the calculated dosages by approx. 3%.

Dose / conc.:

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

Remarks:

The batch no. 0001026602 used in this study had a different concentration of the active substance Thionocarbamate than stated in the original certificate of analysis: GLP-Certificate of October 4th 2012 with 95.7% purity instead of 92.9% stated earlier (Certificate of June 6th 2012). Based on a purity of 92.9%, a correction factor of 1.08 was used in this study. Consequently, the actually administered dosages for the groups were slightly higher (31, 103 and 309 mg IPETC (DANAFLOATTM 262)/kg b.w./day) than the calculated dosages by approx. 3%.

Dose / conc.:

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

Remarks:

The batch no. 0001026602 used in this study had a different concentration of the active substance Thionocarbamate than stated in the original certificate of analysis: GLP-Certificate of October 4th 2012 with 95.7% purity instead of 92.9% stated earlier (Certificate of June 6th 2012). Based on a purity of 92.9%, a correction factor of 1.08 was used in this study. Consequently, the actually administered dosages for the groups were slightly higher (31, 103 and 309 mg IPETC (DANAFLOATTM 262)/kg b.w./day) than the calculated dosages by approx. 3%.

No. of animals per sex per dose:

80 animals (40 male and 40 female rats),
10 animals/sex/group.
A sufficient number to grant at least 8 pregnant
females per group for evaluation of the F0 generation

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: See supporting study: 14-DAY DOSE-RANGE-FINDING STUDY OF IPETC (DANAFLOATTM 262) BY ORAL ADMINISTRATION TO RATS

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily, the frequency was increased when signs of toxicity were observed. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily, The frequency was increased when signs of toxicity were observed. Additionally, once before the first exposure (to allow within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory
pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. selfmutilation, walking backwards) were also recorded. Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at termination of the study. During gestation, females were weighed on days 0, 7,
14 and 20 and within 24 hours of parturition (day 1 post-partum) and day 4 postpartum. Body weights were recorded individually for each adult animal.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: Pre-mating, mating and post-mating period
None of the male animals treated with 31 mg IPETC)/kg b.w./day revealed any test item-related signs of systemic toxicity. Treatment with 103 mg IPETC/kg b.w./day caused reduced motility in all 10 male rats on each day of the first week of the pre-mating period, starting between 5 and 20 minutes after administration and lasting for 2 to 6 hours. This symptom was of moderate degree on test days 1 to 4 and of slight degree on test days 5 to 7. Furthermore, slightly reduced motility was observed in one male (no. 48) on three days (test days 15 to 17) during the mating period.
In addition, increased salivation was observed in one male animal (no. 47) on test days 13 and 14 (moderate degree) during the pre-mating period, on test days 17 and 18 (slight degree) and on test days 39 to 42 (moderate degree) during the mating and post-mating period. This symptom started within 5 minutes after administration and lasted for 20 to 60 minutes. At 309 mg IPETC /kg b.w./day, prone position was seen in all high dose males during test days 1 to 3 (pre-mating period). This symptom was observed on one day (1 of 10 males), two days (3 of 10 males) or three days (6 of 10 males), starting within 5 to 20 minutes after administration and predominantly
lasting for 6 to 24 hours.
Furthermore, reduced motility was daily noted in all high dose male rats from day 3 or 4 onwards until day 21 or 22, starting within 5 to 60 minutes after administration and lasting for 20 minutes up to 6 hours. This symptom was of moderate degree on test days 3 and/or 4, and of slight degree from test day 5 onwards. In addition, increased intake of drinking water and increased salivation were noted in all high dose male animals on several to all test days from test day 7 to 12 onwards until end of test. Salivation started immediately to 20 minutes after administration and lasted for 20 to 60 minutes, being of slight or moderate degree, extremely increased salivation was noted in animal no. 68 on test days 15 to 18. Piloerection was only noted in one male animal (no. 65) on test day 1.

Females: Pre-mating and mating period
None of the female animals treated with 31 mg IPETC /kg b.w./day revealed any test item-related signs of systemic toxicity. Treatment with 103 mg IPETC /kg b.w./day caused reduced motility in all female rats on test days 1 to 7 of the pre-mating period, starting between 5 and 20 min after administration and lasting for 2 to 6 hours. This symptom was of moderate degree on test days 1 to 4 and of slight degree on test days 5 to 7. In addition, piloerection was observed in one intermediate dose female animal (no.57) on test day 1. Furthermore, slightly increased salivation was observed in one intermediate dose female animal (no. 59) on test days 16 to 18 (mating period), starting within 5 minutes after administration and lasting for 20 to 60 minutes. At 309 mg IPETC /kg b.w./day, prone position was seen in all high dose females during test days 1 to 4 of the pre-mating period. This symptom was observed on all 4 days in the 9 surviving females and on 2 days before premature death of female no. 79 on test day 3. Prone position started within 5 to
20 minutes after administration and lasted for up to 6 or up to 24 hours. In addition, piloerection was observed in 7 of 9 surviving high dose female rats (nos. 71, 72, 73, 74, 76, 77 and 80) from test day 1, 2, 3 or 8 onwards. This symptom was noted on one to several test days of the pre-mating period and in one female (no. 72) for totally 16 test days during the pre-mating and mating period. Furthermore, reduced motility was noted in all surviving high dose female rats daily from day 5 onwards until mating, starting within 5 to 20 minutes after administration and lasting for 1 to 6 hours and for up to 24 hours on individual test days. This symptom was of predominantly of slight degree, sometimes of moderate degree. In addition, increased intake of drinking water was noted in 5 high dose females (nos. 71, 73, 76, 77 and 78) during the pre-mating and/or mating period. Apart from the afore-mentioned findings, the following clinical symptoms were only noted in one high dose female (no. 72): haemorrhagic left eye (test days 8 to 10), pultaceous faeces (test day 10), decreased intake of drinking water (test days 10 to 13) and slightly or moderately increased salivation (several days during the mating period), salivation starting immediately to 20 minutes after administration and lasting for 20 to 60 minutes.
Females: Gestation period
None of the female animals treated with 31 mg IPETC /kg b.w./day revealed any test item-related signs of systemic toxicity during the gestation period. At 103 mg IPETC /kg b.w./day, slightly or moderately increased salivation was still noted in one female (no. 59) on a few days and in one female (no. 58) on one day of the gestation period, starting immediately to 20 minutes after administration and lasting for 20 to 60 minutes. A haemorrhagic vagina was noted in one intermediate dose female (no. 51) on gestation day 17. At 309 mg IPETC /kg b.w./day, slightly reduced motility was still noted in 7 of 9 females on 3 to 8 test days of the gestation period (days 0 to 8) starting within 5 to 20 minutes after administration and lasting for 1 to 6 hours and for up to 24 hours on individual test days. In addition, slightly increased salivation was noted in 6 high dose females (nos. 71, 72, 73, 74, 76 and 78) on one to several days of the gestation period, starting immediately to 20 minutes after administration and lasting for 20 to 60 minutes. Increased intake of drinking water was noted in 5 high dose females (nos. 71, 72, 76, 77 and 78) on several to all gestation days. One high dose female (no. 78) revealed piloerection on 2 days of the gestation period.
Females: Lactation period
Piloerection was noted in 2 of 10 dams (nos. 36 and 38) treated with 31 mg IPETC /kg b.w./day on 1 or 2 days during the lactation period. At 103 mg IPETC /kg b.w./day, only 3 of 10 dams littered as a total post-implantation loss was noted in the remaining 7 dams. None of these 3 female animals revealed any test item-related signs of systemic toxicity during the lactation period.
No lactation period was determined for the high dose level as administration of 309 mg IPETC /kg b.w./day caused a total post-implantation loss in all treated dams.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Males: No deaths were noted in the male rats treated with 31, 103 or 309 mg IPETC /kg b.w./day.
Females: No deaths occurred during treatment of the female rats with 31, 103 or 309 mg IPETC /kg b.w./day except for one high dose dam (no. 79) which was found dead on test day 3. Prone position was observed on the days before death, necropsy revealed gastric lesions. The death of this animal is considered as test item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals: Pre-mating, mating and post-mating period
No test item-related influence was noted on the body weight or the body weight gain of the male animals treated with 31 mg IPETC kg b.w./day during the pre-mating, mating and post-mating period. The mean body weight gain from the start of treatment (test day 1) was plus 76.0% (control: plus 76.2%) at the end of treatment (test day 43). At 103 mg IPETC kg b.w./day, a marginal, not statistically significant reduction in body weight (5% below the control value) was noted at the end of the study on test day 43. The mean body weight gain from the start of
treatment (test day 1) was plus 67.0% (control: plus 76.2%) at the end of treatment (test day 43). The differences are regarded to be spontaneous. The body weight of the high dose males (309 mg IPETC kg b.w./day) was reduced from test day 8 until end of the study on test day 43 (p ≤ 0.01), being approx. 9 to 12% below the control values. Body weight and body weight gain were statistically significantly (p ≤ 0.01) reduced on test days 8, 15, 22, 29, 36 and 43. The mean body weight gain from the start of treatment (test day 1) was plus 55.3% (control: plus 76.2%) at the end of treatment (test day 43).

Female animals: pre-mating period
No test item-related influence was noted on the mean body weight and the body weight gain during the pre-mating period (test day 1 to test day 15) at the low dose level (31 mg IPETC /kg b.w./day). The mean body weight gain from the start of treatment (test day 1) was plus 8.4%, respectively (control: plus 10.0%) at the end of the pre-mating period (test day 15). At 103 mg IPETC /kg b.w./day, slight reductions were noted for the body weight (up to 7% below the control values) during pre-mating period. Statistically significant reductions compared to the control were noted in the first test week for the body weight (at p ≤ 0.05) and the body weight gain (at p ≤ 0.01). The mean body weight gain from the start of treatment (test day 1) was plus 3.1% (control: plus 10.0%) at the end of the pre-mating period (test day 15). Statistically significant reductions at p ≤ 0.01 were noted for the body weight of the high dose females (309 mg IPETC /kg b.w./day) during
the first and second test week of the pre-mating period (up to 12% below the control values). Statistically significant reductions compared to the control (at p ≤ 0.01) were accordingly noted for the body weight gain. A negative body weight gain (3.9%, control: plus 10.0%) was noted from the start of treatment (test day 1) until end of the pre-mating period (test day 15).
Female animals: gestation period
No test item-related influence was noted on the body weight and body weight gain of the female animals treated with 31 mg IPETC /kg b.w./day during the gestation period. The increase in the mean body weight from day 0 of gestation until day 20 was plus 54.0% (control: plus 59.4%).
At 103 mg IPETC /kg b.w./day, statistically significant reductions (at p ≤ 0.01) were noted for the body weight on gestation days 14 and 20 (up to 26.4% below the control values). The body weight gain was accordingly reduced (significant at p ≤ 0.01). The mean body weight gain from the start of treatment (test day 1) was only plus 18.9% (control: plus 59.4%) on gestation day 20. At 309 mg IPETC /kg b.w./day, statistically significant reductions (at p ≤ 0.01) were noted for the body weight on gestation days 7, 14 and 20 (up to 33.3% below the control values). The body weight gain was significantly reduced (at p ≤ 0.05 or p ≤ 0.01) on gestation days 7, 14 and 20. The mean body weight gain from the start of treatment (test day 1) was only plus 12.8% (control: plus 59.4%) on gestation day 20. The absence of the expected body weight increase during the gestation period
was obviously due to the high incidences of total post-implantation loss precluding the typical body weight increase during pregnancy at 103 mg IPETC/ kg b.w./day (7 of 10 females were affected) and 309 mg IPETC /kg b.w./day (all 9 surviving females were affected).

Female animals: lactation period
No test item-related influence was noted on the body weight and body weight gain of the females treated with 31 mg IPETC / kg b.w./day (10 littering dams) or with 103 mg IPETC /kg b.w./day (3 littering dams) during the lactation period. No body weight was recorded for the high dose females during the lactation period as none of these dams littered due to a total post-implantation loss.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: pre-mating period
No test item-related effect on food consumption was noted in the male rats treated with 31 or 103 mg IPETC /kg b.w./day during the pre-mating period. At 309 mg IPETC /kg b.w./day, a statistically significant reduction (p ≤ 0.01) by 15.4% compared to the control was noted for the relative food intake of the male rats in the first test week of the pre-mating period. The relative food intake was statistically significantly increased (at p ≤ 0.01) compared to the control in the second test week. This increase has to be assessed by considering the reduced body weight noted for the high dose males at this time as the absolute food consumption was not increased.

No food intake of female animals was recorded during the mating period as both sexes were housed together.

Females: gestation period
No test item-related influence on food consumption was found in the female rats after treatment with 31 or 103 mg IPETC /kg b.w./day during the gestation period. At 309 mg IPETC /kg b.w./day, the relative food intake was marginally reduced (up to 11.6% below the control) during the gestation period.
Females: lactation period
At 31 or 103 mg IPETC /kg b.w./day, the relative food intake was marginally reduced (14.7% below the control for the low dose group and 5.9% below the control for the intermediate dose group) during the lactation period. No food intake was recorded for the high dose females during the lactation period as none of these dams littered due to a total post-implantation loss.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male and female animals:
No test item-related influence was noted in the haematological parameters examined at the end of the pre-mating period (test day 15) of male and female rats treated with 31, 103 or 309 mg IPETC /kg b.w./day. No test item-related influence was noted for the haemoglobin content, the number of erythrocytes, leucocytes, reticulocytes and platelets, the haematocrit value, the thromboplastin time and the activated partial thromboplastin time, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC) compared to the control. No test item-related changes were noted in the relative and absolute differential blood count.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female animals:
No test item-related changes were noted on biochemical parameters on test day 15 for male and female rats treated with 31 or 103 mg IPETC /kg b.w./day compared to the control. This includes also the serum level of bile acids of the male rats of the low and intermediate groups except for one outlier in the low dose group. Bile acids are known to show a high inter-individual variability. In addition, the sampling size of n = 5 animals per sex is fairly small (see table below). At 309 mg IPETC /kg b.w./day, test item-related and statistically significantly (p≤0.01) increased mean values were obtained for the plasma levels of cholesterol (total) of the male (by 26%) and female (by 68%) rats and for the serum levels of the bile acids (by 984%) of the male rats.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Examination of gestation length
Note: The calculation of the gestation length includes the day of positive sperm detection (mating day or gestation day 0) and the day/s of littering. The day, on which there was no sign of littering anymore in the morning is defined as litter day 1 and is not part of the gestation period anymore.
No test item-related influence was noted on the gestation length of the females treated with 31 mg IPETC /kg b.w./day compared to the control group. A total post-implantation loss was noted in 7 of 10 females treated with 103 mg IPETC /kg b.w./day. The gestation length of the remaining
3 females were within the normal range. No data on gestation length were available for the high dose dams (treated with 309 mg IPETC /kg b.w./day) as a total post-implantation loss was noted in all 9 surviving dams.

Evaluation of reproduction parameters of F0 dams
Corpora lutea and implantation sites. No statistically significant differences were noted in the number of corpora lutea or implantation sites between the control and the treatment groups (31, 103 or 309 mg IPETC /kg b.w./day). No test item-related increases were noted for the pre-implantation loss at any tested dose level. Number of pups No female aborted. No runts or malformed pups were noted at birth. At 31 mg IPETC /kg b.w./day, the number of born pups (alive
and dead) was within the normal range. However, statistically significant reductions (at p ≤ 0.01) were noted for the birth index (calculated from the total number of born pups and the total number of implantation sites), being 76% (control: 97%). This reduction in the birth index was considered as test item related. The live birth index was slightly reduced to 95.9% in comparison to the control with 99.2%. This slight reduction was considered as not test item related. In detail, the number of pups born alive was only marginally reduced due to the incidence of 5 stillbirths. Stillbirths were noted in dam no. 32 (two stillbirths) and in dam no. 39 (three stillbirths). One stillbirth was noted in the control group (dam no. 19). The gestation index of 90% (control: 100%) was still within the range of LPT background data (see table below). No living pups, only two stillbirths were noted in one of 10 litters (dam no. 32). A statistically significant increase (at p ≤ 0.01) was noted for the post implantation loss (26.9%, control: 3.6%; for LPT background data see table below), which was considered as test item related. At 103 mg IPETC /kg b.w./day), a statistically significant increase (at p ≤ 0.01) was noted for the post-implantation loss (96.4%, control: 3.6%) caused by a complete loss of implants in 7 of 10 examined females. Statistically significant reductions (at p ≤ 0.01) were noted for the gestation index (30%, control: 100%), the birth index (4%, control: 97%), the number of born pups (alive and dead) and the number of live born pups. In total, only 5 pups (all of them live born) were found in 3 examined litters: one
pup each in the litter of dams no. 52 and 53, and 3 pups in the litter of dam no. 56. Hence, a live birth index of 100% was calculated (control: 99.2%; for LPT background data see table below).
No data on pups were available for the high dose dams (treated with 309 mg IPETC /kg b.w./day) as a total post-implantation loss was noted in all 9 surviving dams (post-implantation loss: 100%, control: 3.6%). Statistically significant reductions (at p ≤ 0.01) were noted for the gestation index
(0%, control: 100%) and the birth index (0%, control: 97%)

Key result

Dose descriptor:

NOAEL

Effect level:

31 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

for effects on reproductive toxicity

Effect level:

< 31 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Effects on reproductive toxicity NOAEL (no-observed-adverse-effect level): below 30 mg/kg b.w./day

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No statistically significant differences were noted between the survival index (viability
index) of the control group and the low and intermediate dose groups (31 or 103 mg IPETC /kg b.w./day). One stillbirth was noted in the control group (dam no. 19). Three control pups from two different dams (nos. 16 and 19) were cannibalized on lactation day 3 or 4, leading to a mean survival index of 98.0%, hence, no effect was observed. In the low dose group (31 mg IPETC /kg b.w./day), five stillbirths were noted. No living pups, only two stillbirths were noted in one of 10 litters (dam no. 32). Three stillbirths were found in the litter of dam no. 39. In addition, two pups from two dams (nos. 34 and 39) were cannibalized on lactation day 3 or 4, leading to a total viability index of 98.3% (control: 97.7%). In the intermediate dose group (103 mg IPETC /kg b.w./day), only 5 pups (all of them live born) were found in 3 examined litters. One pup (dam no. 53) was found dead one day after birth, leading to a total viability index of 80% (control: 97.7%) and a mean viability index of 66.7% (98.0%) on gestation day 4. A second pup (dam no. 52) was found dead before sacrifice on lactation day 5. No data on pups were available for the high dose dams (treated with 309 mg
IPETC /kg b.w./day) as a total post-implantation loss was noted in all 9 surviving dams

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight
At 31 mg IPETC /kg b.w./day, a slight - not statistically significant - reduction in mean litter weight was noted on lactation day 1 for male pups, female pups and total pups (approx. 6% below the control value). On lactation day 4, the mean litter weight was within the range of the control.
In the intermediate dose group (103 mg IPETC /kg b.w./day), normal weights were for the mean litter weight on lactation day 1 calculated for only five pups available in this group. Reduced mean litter weights - not statistically significant compared to the control - were calculated on lactation day 4 for the two male pups (approx. 16% below the control) and for the total of four surviving
pups. This finding was due to the reduced body weight noted for the single male pup of dam no. 52. Total litter weight
No statistically significant differences were noted between the total litter weight of the pups at 31 mg IPETC /kg b.w./day or the control pups. A statistically significant reduction (at p ≤ 0.01) in total litter weight was noted for the intermediate dose group (103 mg IPETC /kg b.w./day) on lactation days 1 and 4 (male and total pups). Due to the low incidence of only 2 female pups no statistical comparison could be carried out for the female pups. No data on mean or total litter weights were available for the high dose dams (treated with 309 mg IPETC /kg b.w./day) as a total ostimplantation loss was noted in all 9 surviving dams.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

ca. 31 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Reproductive effects observed:

not specified

Conclusions:

Treatment with IPETC produced significant post-implantation loss (early abortions), with a very steep dose-response. Grp mean values were: 3.6% (control), 26.9%, 96.4%, and 100%. Based on the (limited) investigations in this screening study, the effect appears to be very specific, and did not correlate with parental toxicity. Therefore, IPETC appear to be a developmental toxicant. No NOAEL for the offspring could be established from this study, but it can be concluded that it lies below 31 mg/kg bw./day, which is this studies LOAEL.

Executive summary:

Toxicity to reproduction test was done with OECD Guideline 422. Crl:CD rats were exposed to test item for 28days. test item was given by oral gavage once a daily. For parental animals clinical signs, mortality and body weight changes were observed. NOAEL is determined to be 31 mg/kg bw/day. For pups viability, mortality and body weight changes were observed. LOAEL is determined to be 31mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

One GLP study available with Klimisch score 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction test was done with OECD Guideline 422. Crl:CD rats were exposed to test item for 28days. The test item was given by oral gavage once daily. For parental animals clinical signs, mortality and body weight changes were observed. NOAEL is determined to be 31 mg/kg bw/day. For pups viability, mortality and body weight changes were observed. LOAEL is determined to be 31 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Developmental toxicity: OECD 414; Wistar pregnant female rats; NOAEL for the maternal toxicity: 30 mg/ kg body weight/ day; NOAEL for the embryo-fetal developmental toxicity: 10 mg/ kg body weight/ day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016 - 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Cheminova A/S Batch No. 0001026602
- Expiration date of the lot/batch: October 13th, 2017
- Purity test date: October 13th , 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle:Soluable and stable in the vehicle

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females : approx. 11-12 weeks old, males:Age of the males at the start of pairing: between 12 weeks and not older than 17 weeks
- Weight at study initiation: Body weight before initiation of pairing: males: 320 – 350 g (mean: 334.40 g, ± 20% = 267.52 – 401.28 g) females: 203 - 242 g (mean: 222.76 g, ± 20% = 178.21 – 267.31 g)
- Fasting period before study:
- Housing: Full barrier in an air-conditioned room. The animals were kept individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male)
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum):Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period:t 5-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17th February 2016 To:17th June 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 0.78, 2.6 and 7.8 mg/l
- Amount of vehicle (if gavage): 4 ml/kg bw/day
- Lot/batch no. (if required): Manufacturer: Sigma, Batch No.: MKBS6944V

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: 1-6 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear; referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

20 days

Frequency of treatment:

daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control group (group C)

Dose / conc.:

3 mg/kg bw/day

Remarks:

Group name LD.
Purity level of 95,7 w/w % of test item solution taking into consideration, so dosage concentration is in mg AI/ kg bw /day

Dose / conc.:

10 mg/kg bw/day

Remarks:

Group name MD.
Purity level of 95,7 w/w % of test item solution taking into consideration, so dosage concentration is in mg AI/ kg bw /day

Dose / conc.:

30 mg/kg bw/day

Remarks:

Group name HD.
Purity level of 95,7 w/w % of test item solution taking into consideration, so dosage concentration is in mg AI/ kg bw /day

No. of animals per sex per dose:

24-25 females per dose (males were not exposed, but removed from cages after positive vaginal smear

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, except on weekends and public holidays when observations were made once daily
- Cage side observations included: morbidity and mortality, spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20% variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, although this was not a feeding study; Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
On gestation day 20, sperm positive females were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally at a dosage of approximately 8 mL/kg bw.
At the time of termination, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes, which may have influenced the pregnancy. Unusual growth in fat tissue attached to the uterus (with oviduct and cervix) of female no. 60 of MD group was preserved in 4% neutral-buffered formaldehyde for potential histopathological examination.
Immediately after the termination, the uteri were removed, and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed. The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or fetal deaths as well as the number of viable fetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of fetuses in each uterine horn was also recorded.
Males were used for other studies.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
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 ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter]

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse clinical signs in the animals of the control and test-tem treated groups. Alopecia (on various parts- hindlimb, forelimb, abdomen and dorsal back of the body) was noted in a few animals of control and dose groups. There was no dose-response relationship for this finding and it was considered spontaneous.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on body weight and body weight gain. Slight, statistically significantly higher mean body weights were noted in LD and HD groups on GD 0, 5, 8 and 11 and also on GD 14 in LD group. These changes were not dose-dependent and as noted already prior to dosing not considered test item related. The mean body weights on GD 17 and GD 20 in dose groups were comparable to the corresponding control group. No statistically significant changes in body weight gain were noted between treated and control groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not affected in dose group animals when compared to control group. No statistically significant differences were noted between the dose and control groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy, macroscopic findings relevant to test item treatment were not observed. One female (no. 60) of MD group had a high amount of fat tissue attached to the uterus (with oviduct and cervix). This finding was spontaneous in nature as the finding occurred in a single isolated female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically, no significant differences noted in prenatal parameters including uterus weight, the number of corpora lutea, the number of implantation sites, the number of resorptions (early, late and total), the number of live fetuses, the number of dead fetuses, the number of female fetuses, sex ratio, percent pre-implantation and post-implantation losses. A statistically significantly higher number of mean male fetuses was observed in LD group compared to the control, which in the absence of dose-response relation was considered incidental. Higher mean post-implantation losses in MD and HD groups compared to the control, were in the absence of dose-response relationship and absence of statistical significance were not considered test item related. Further, all mean values for percent pre- and postimplantation loss observed in control and test item treated groups were within the range of historical control data (0 to 51.20 % and 0 to 32.15 %, respectively).
There was a single dead fetus in female no. 100 of HD group, which was an isolated incident and was considered incidental.
Slightly higher numbers of early and total resorptions were noted in the HD group compared to the control group. However, the values in test-item groups were not statistically significantly different compared to the control and the mean values were also within the range of the historical control data (0 to 2.7 %). Therefore, the findings were not considered an adverse effect of test item treatment.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

act. ingr.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Localisation:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal weight in the HD group was statistically significantly lower compared to the control group value (3.38 g in HD Vs 3.70 g in C). However, the numbers of live fetuses and total litter weight were not statistically different in HD group compared to the control. Further, the mean fetal weight in HD group was within the range of historical control data (2.63 g to 4.71 g). However in light of other findings potentially indicative of embryotoxicity (delayed ossification of bones) a test item-related effect on fetal weights in the HD group cannot be ruled out.
The mean male litter weight in the LD group was statistically significantly higher compared to the control value (26.23 g in LD Vs 20.80 g in C). In the absence of a dose- response relationship, this finding was considered incidental.
There were no statistically significant differences noted for total litter weight and female litter weight of treated groups compared to the corresponding control.
There were comparable numbers of fetuses in both (left and right) uterine horns of control and test item treated groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean fetal weight in the HD group was statistically significantly lower compared to the control group value (3.38 g in HD Vs 3.70 g in C). However, the numbers of live fetuses and total litter weight were not statistically different in HD group compared to the control. Further, the mean fetal weight in HD group was within the range of historical control data (2.63 g to 4.71 g). However in light of other findings potentially indicative of embryotoxicity (delayed ossification of bones) a test item-related effect on fetal weights in the HD group cannot be ruled out.
The mean male litter weight in the LD group was statistically significantly higher compared to the control value (26.23 g in LD Vs 20.80 g in C). In the absence of a dose- response relationship, this finding was considered incidental.
There were no statistically significant differences noted for total litter weight and female litter weight of treated groups compared to the corresponding control.
There were comparable numbers of fetuses in both (left and right) uterine horns of control and test item treated groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Hematoma was noted in few isolated fetuses from all test item treated and control groups without any dose- response relationship. This finding was concluded as not related to treatment with the test item. Malformations were noted in two fetuses of the HD group. Fetus No. 7 (dam no. 81) had an absent left eye and misshapen snout. Fetus no. 5 (dam no. 82) had an umbilical hernia. However, these malformations occurred in single isolated fetuses of the HD group and additionally, the finding absent eye was within the historical control range. Therefore, these findings were considered spontaneous in origin. One fetus of the control group was noted with a malformation (right hindlimb rotated).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The fetal skeletal examinations found a statistically significantly increased incidence of findings in the HD group, including incomplete ossification of skull frontal bone (bilateral) (35% in HD and 0% in C), zygomatic arch (right) (35% in HD and 5% in C) and supraoccipital bone (80% in HD and 35% in C); statistically significantly higher incidence of unossified metacarpals (60% in HD and 10% in C); statistically significantly higher incidences of rudimentary right cervical rib (7th) (25% in HD and 0% in C); statistically significantly higher incidences of wavy ribs (80% in HD and 45% in C); and a statistically significantly higher incidence of misshapen humerus (25% in HD and 0% in C). In addition, a statistically significantly increased incidence of incomplete ossification of parietal bone (bilateral) was noted in the in MD group (65% in MD and 25% in C), and a statistically significantly increased incidence of incomplete ossification of pelvic girdle pubis in LD group (23% in LD and 0% in C).
Moreover, increased incidences of incomplete ossification of frontal (bilateral) (20% in MD group and 0% in C) , hyoid body (25% in HD and 5% in C), interparietal (75% in MD, 85% in HD and 55% in C), bent scapula and spine (right sided) (15% in HD and 0% in C), bent scapula (right sided and bilateral) (25% and 20% in HD and 5% and 0% in C, respectively). These findings did not attain the statistical significance compared to the corresponding control.
When compared to historical control data (HCD), the majority of these skeletal findings occurred at a higher incidence than HCD mean value, but for most observations within HCD ranges. However, there were incidences which were either outside the range of HCD or the HCD incidence was 0.

The findings of incomplete ossification of various bones, including frontal, zygomatic, supraoccipital, metacarpals and hyoid body are variations associated with delayed ossification and transient in nature. Also wavy ribs are conformational bone changes that are transient and reversible [11] [17] and of minor toxicological significance. In combination with the observed lower fetal weight these findings are considered a sign of test item related embryotoxicity in the HD group. In the absence of associated signs of adverse effects, the increased incidence of incomplete ossification of parietal bone, rudiementary 14th rib, bent scapula and long bones observed in the MD group is not considered an adverse effect of the test item [17]. The increased incidence of incomplete ossification of pelvic girdle pubis in the LD group did not show dose-response relationship and was therefore concluded not to be related to treatment with the test item. The other skeletal findings listed in the above table were statistically not significantly different compared to the corresponding control and/or were not dose response related.

The statistically significantly higher incidences of rudimentary cervical rib (7th) in HD group are considered adverse effects of treatment with the test item because the cervical ribs causes considerable adverse effects in human [11], but in rats it is a minor variation [16]. The misshapened bones represent malformations, irrespective of the structure concerned [11], thus the statistically significantly higher incidences of misshapen humeri in HD group was an adverse effect of test-item treatment.

Visceral malformations:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

act. ingr.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: forelimb

skeletal: rib

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with IPETC at dose levels of 3, 10, and 30 mg/ kg body weight/ day administered on gestation days 5 to 19, the following conclusions can be made: There were no test item related findings in maternal animals.
There were increased incidences of rudimentary cervical rib (7th) and misshapen humerus in high dose (HD) group compared to the corresponding control and were considered to be adverse effects. In addition, in the same group, increased incidences of reversible skeletal variations and reduced fetal weights were considered signs of test item related embryotoxicity. Thus, the NOAEL for the maternal toxicity is 30 mg/ kg body weight/ day and the NOAEL for the embryo-fetal developmental toxicity is 10 mg/ kg body weight/ day.

Executive summary:

Developmental toxicity test was done according to OECD Guideline 414. Wistar pregnant female rats were exposed to test item concentration of 3, 10 and 30mg/kg bw/day for 20 days. There were no test item related findings in maternal animals. There were increased incidences of rudimentary cervical rib (7th) and misshapen humerus in high dose (HD) group compared to the corresponding control and were considered to be adverse effects. In addition, in the same group, increased incidences of reversible skeletal variations and reduced fetal weights were considered signs of test item related embryotoxicity. Thus, the NOAEL for the maternal toxicity is 30 mg/ kg body weight/ day and the NOAEL for the embryo-fetal developmental toxicity is 10 mg/ kg body weight/ day.

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:

Guideline study

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

Developmental toxicity test was done according to OECD Guideline 414. Wistar pregnant female rats were exposed to test item concentration of 3, 10 and 30mg/kg bw/day for 20 days. There were no test item related findings in maternal animals. There were increased incidences of rudimentary cervical rib (7th) and misshapen humerus in high dose (HD) group compared to the corresponding control and were considered to be adverse effects. In addition, in the same group, increased incidences of reversible skeletal variations and reduced fetal weights were considered signs of test item related embryotoxicity. Thus, the NOAEL for the maternal toxicity is 30 mg/ kg body weight/ day and the NOAEL for the embryo-fetal developmental toxicity is 10 mg/ kg body weight/ day.

Justification for classification or non-classification

A recent OECD 422 study, conducted in the context of this REACH registration, has shown result in developmental toxicity indicating that the substance lives up to the classification criterion of the CLP regulation 1272/2008 as toxic to reproduction in category 2 or maybe even category 1B. OECD 422 is a screening study usable for classification and its limitations as grounds for classification is addressed in the CLP regulation, in Annex 1, Part 3, section 3.7.2.5.2. A further OECD 414 study addresses these previous findings of signs of developmental toxicity to the point where a precise classification can be determined, and the substance is classified as a reproductive toxicant Cat. 2 (H361) in accordance with article 37 of Regulation (EC) No 1272/2008.