2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In accordance with column 2 of REACH annex VIII, screening for reproductive/developmental toxicity does not need to be conducted if a pre-natal developmental toxicity study is available.

 

In accordance with section 8.7.3 of Columns 1 and 2 of REACH Annexes IX and X, it is considered that the extended one generation reproductive toxicity study (EOGRTS) is not required.  The EOGRTS is not proposed at this time as it is considered that DMTE does not meet the criteria for conducting it. This is based on the recent EU Commission Regulation 2015/282 (February 20, 2015) which amended EC 1907/2006 Article IX section 8.7.3. The EU Expert Group recommended that an exposure-based trigger be adopted for substances which have uses leading to significant exposures of consumers and professionals.  DMTE does not have uses which lead to significant exposure of professionals or consumers. DMTE is approved for Food Contact use in both the United States and the EU. DMTE is also approved by NSF International for use in PVC pipe. Extraction studies are required for both types of approval and in both cases the estimated worst-case potential for consumer exposures is low.  DMTE does not meet the toxicological criteria in Annex IX/X for the EOGRTS study. The substance is not mutagenic. There is no evidence that DMTE has an adverse effect on male or female reproductive organs or adversely effects fertility. Further, there is clear evidence in animals and humans that an internal dose for methyltin substances is not slow to reach a steady state; it is rapidly excreted in the urine and faeces. The substance is not an endocrine disruptor. Lastly, while an adverse outcome in a teratology study [OECD 414] could be a trigger for the EOGRTS, the results of the OECD 414 for DMTE showed no test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated. Further detail is available in the overall testing strategy document attached to section 13.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Justification for type of information:

In accordance with column 2 of REACH annex VIII, screening for reproductive/developmental toxicity does not need to be conducted if a pre-natal developmental toxicity study is available.

Reproductive effects observed:

not specified

Reference 2

Endpoint:

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

In accordance with section 8.7.3 of Columns 1 and 2 of REACH Annexes IX and X, it is considered that the extended one generation reproductive toxicity study (EOGRTS) is not required. The EOGRTS is not proposed at this time as it is considered that DMTE does not meet the criteria for conducting it. This is based on the recent EU Commission Regulation 2015/282 (February 20, 2015) which amended EC 1907/2006 Article IX section 8.7.3. The EU Expert Group recommended that an exposure-based trigger be adopted for substances which have uses leading to significant exposures of consumers and professionals. DMTE does not have uses which lead to significant exposure of professionals or consumers. DMTE is approved for Food Contact use in both the United States and the EU. DMTE is also approved by NSF International for use in PVC pipe. Extraction studies are required for both types of approval and in both cases the estimated worst-case potential for consumer exposures is low. DMTE does not meet the toxicological criteria in Annex IX/X for the EOGRTS study. The substance is not mutagenic. There is no evidence that DMTE has an adverse effect on male or female reproductive organs or adversely effects fertility. Further, there is clear evidence in animals and humans that an internal dose for methyltin substances is not slow to reach a steady state; it is rapidly excreted in the urine and faeces. The substance is not an endocrine disruptor. Lastly, while an adverse outcome in a teratology study [OECD 414] could be a trigger for the EOGRTS, the results of the OECD 414 for DMTE showed no test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated. Further detail is available in the overall testing strategy document attached to section 13.

Reproductive effects observed:

not specified

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

Effects on developmental toxicity

Description of key information

Key Study: Langedijk (2021)

Under the conditions of the study, the NOAELs for the test material were:

Maternal toxicity: 10 mg/kg/day, based on decreased food consumption and lower mean body weight gain adjusted for gravid uterus.

Developmental toxicity: At least 25 mg/kg/day (no test material-related adverse findings).

 

Supporting Study: Langedijk (2021) Range-finder

Under the conditions of this study, selected dose levels for the subsequent Prenatal Developmental Toxicity study by oral gavage are 3, 10 or 25 mg/kg/day, on the basis that a dose of 50 mg/kg/day was clearly in excess of the maximum tolerated dose being associated with severe body weight loss and low food consumption, necessitating the early termination of the group. The dose level of 25 mg/kg bw/day was associated with clear but tolerable maternal toxicity in terms of clinical signs, reduced body weight and body weight gain, lower food intake, lower absolute and relative thymus weight with macroscopic correlate. Therefore it was considered that a dose of 25 mg/kg/day would adequately demonstrate systemic toxicity in accordance with the OECD 414 test guideline requirements. Dose levels of 3 and 10 mg/kg/day were selected on the expectation they would characterise a dose-response, with the lowest dose expected to be a No Adverse Effect Level (NOAEL).

 

Disregarded Study: Manjunath (2021)

The study was terminated without finalization of study report or interpretation of the data as the data was considered as not valid.

 

Developmental Toxicity Second Species Study

A developmental study has been conducted in the rat according to OECD Test Guideline 414 on DMTE, which showed no test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated. A second species developmental toxicity test in rabbits is proposed only if adverse effects are observed in rats. There is no evidence that the methyltins are teratogenic and a second species test is therefore not warranted.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

A developmental study has been conducted in the rat according to OECD Test Guideline 414 on DMTE, which showed no test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated. A second species developmental toxicity test in rabbits is proposed only if adverse effects are observed in rats. There is no evidence that the methyltins are teratogenic and a second species test is therefore not warranted.

Species:

rabbit

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

24 December 2018 to 13 May 2019

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Details on exposure:

- Dose volume: 10 mL/kg body weight.

Duration of treatment / exposure:

Gestation Day (GD) 5 to 19.

Dose / conc.:

5 mg/kg bw/day

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on maternal toxic effects:

A total of 22, 21, 21 and 22 presumed pregnant female were confirmed as pregnant (with evidence of implantations) across groups, yielding to pregnancy rates of 88 %, 84 %, 84 % and 88 % at the time of caesarean section from the groups G1, G2, G3 and G4, respectively. Among these, a total of 22, 21, 20 and 20 presumed pregnant females from group G1, G2, G3 and G4, respectively were found with live fetuses.
Upon review of the data by the Sponsor, the study was terminated without finalisation of study report or interpretation of the data as the data was considered as not valid by sponsor. All the data generated during the study period will be archived.

Dose descriptor:

NOAEL

Remarks on result:

not determinable because of methodological limitations

Abnormalities:

not specified

Dose descriptor:

NOAEL

Remarks on result:

not determinable because of methodological limitations

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The study was terminated without finalisation of study report or interpretation of the data as the data was considered as not valid

Executive summary:

The objective of this study was to assess the effects of exposure to the test material on the presumed pregnant rats and in the developing organisms, including an assessment of maternal effects as well as death, structural abnormalities, or altered growth in the foetus.

A total of 100 presumed pregnant female Sprague Dawley rats were allocated to four groups. Each group consisted of 25 presumed-pregnant female rats (Dams). The test material was administered by oral gavage, at a dose volume of 10 mL/kg body weight to each animal, with the doses of 0, 5, 15 and 50 mg/kg body weight for groups G1 (Vehicle Control), G2 (Low Dose), G3 (Mid Dose) and G4 (High Dose), respectively from Gestation Day (GD) 5 to 19.

A total of 22, 21, 21 and 22 presumed pregnant female were confirmed as pregnant (with evidence of implantations) across groups, yielding to pregnancy rates of 88 %, 84 %, 84 % and 88 % at the time of caesarean section from the groups G1, G2, G3 and G4, respectively. Among these, a total of 22, 21, 20 and 20 presumed pregnant females from group G1, G2, G3 and G4, respectively were found with live fetuses.

Upon review of the data by the Sponsor, the study was terminated without finalisation of study report or interpretation of the data as the data was considered as not valid by sponsor.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 February 2021 to 10 March 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this dose range finding study, the test material was administered to pregnant Wistar Han rats from Day 6 up to and including Day 20 post-coitum by oral gavage at 25 or 50 mg/kg bw/day. At intervals throughout the day, mortality and cage side clinical observations were recorded. Every third day during treatment and on the day of necropsy, detailed clinical observations, body weights, food consumption were recorded.

GLP compliance:

no

Remarks:

GLP compliance is not claimed for this range-finding study.

Limit test:

no

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Adjustment will be made for specific gravity of the test material and vehicle. No correction will be made or the purity/composition of the test material.

Species:

rat

Strain:

Wistar

Remarks:

Rat, Crl: WI(Han), Outbred, SPF-Quality.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 - 15 weeks old at initiation of dosing.
- Weight at study initiation: 202 - 244 g at start of dosing.
- Housing: Individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: Ad libitum access to pelleted rodent diet.

ENVIRONMENTAL CONDITIONS
- Temperature: The actual daily mean temperature during the study period was 22 °C.
- Humidity: The actual daily mean relative humidity was 43 to 50 %.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly, filled out in daily portions and stored in the refrigerator protected from light. Dosing formulations were removed from the refrigerator at least 30 minutes before dosing and were stirred at room temperature. The dosing formulations were kept at room temperature until dosing. The dosing formulations were continuously stirred until (if practically possible) and during dosing. Any residual volumes were discarded. Dose formulation analysis for concentration, stability or homogeneity was not performed, however, to limit the impact, the test material preparation was performed with approved procedures as demonstrated previously in the analytical method development and validation study and documented in detail. Formulations were visually inspected for homogeneity prior to use.
- Dose Volume: 5 mL/kg. The dose volume for each animal will be based on the most recent body weight measurement.
- Dose concentration:
25 mg/kg bw/day: 5 mg/mL
50 mg/kg bw/day: 10 mg/mL

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant. Day 0 post-coitum is the day of successful mating.

Duration of treatment / exposure:

Day 6 up to and including Day 20 post-coitum.

Frequency of treatment:

Daily, 7 days a week.

Duration of test:

Group 1: From Day 6 to Day 21 post-coitum.
Group 2: From Day 6 to Day 15 post-coitum (early termination).

Dose / conc.:

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

Remarks:

Group 1

Dose / conc.:

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

Remarks:

Group 2

No. of animals per sex per dose:

Six females per dose.

Control animals:

yes, historical

Details on study design:

- Dose selection rationale: The OECD 414 guideline details that the highest dose should induce some developmental/maternal toxicity without inducing suffering or death. It is expected, based on available toxicity data, that a dose level of 50 mg/kg bw/day will induce maternal toxicity in terms of overall reduction in bodyweight gain (not loss). To inform on dose response, a low dose of 25 mg/kg bw/day is recommended.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At intervals throughout the day, mortality and cage side clinical observations were recorded.
Mortality: Twice daily throughout the study.
Cage Side Clinical Observations: At least once daily starting on Day 6 post-coitum up to the day prior to necropsy. During the dosing period, this observation will be performed 1 hour after dosing.
Cage debris will be examined to detect premature birth, if applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At intervals throughout the day, cage side clinical observations were recorded. Every third day during treatment and on the day of necropsy, detailed clinical observations were recorded.
Detailed Clinical Observations: Prior to dosing Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.
Animals showing pain, distress or discomfort (as was noted for Group 2) which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of these deaths were recorded in detail.

BODY WEIGHT: Yes
- Time schedule for examinations: Every third day during treatment and on the day of necropsy body weights were recorded. Days 2, 6, 9, 12, 15, 18 and 21 post-coitum (prior to dosing, if applicable).
Body weight gain will be calculated over each sequent interval and over the total study period. In addition, body weight gain will be calculated over Days 6 - 15 post-coitum. Corrected body weight gain will be determined based on Day 6 and Day 21 post-coitum body weights and the gravid uterus weight.

FOOD CONSUMPTION: Yes
- Time schedule: Every third day during treatment and on the day of necropsy, food consumption was recorded. Days 2 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post-coitum.

WATER CONSUMPTION: No.

POST-MORTEM EXAMINATIONS: Yes. Animals will be euthanised using the carbon dioxide inhalation (gradual fill) procedure.
- Sacrifice on gestation day:
Group 1: Day 21 post-coitum (scheduled termination).
Group 2: Day 15 post-coitum (early termination).
- Organs examined: External, thoracic and abdominal examination, with special attention being paid to the reproductive organs. Number of corpora lutea, weight of the liver, thymus and uterus (not for animals found dead, sacrificed before planned necropsy or that delivered early), placental weights of live foetuses, number of implantation sites, number and distribution of live and dead foetuses, number and distribution of early and late resorptions were determined. Macroscopic abnormalities were recorded, collected and fixed.
All macroscopic abnormalities will be recorded, collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution). The liver and thymus will not be retained.

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 examination, external sex determination and body weight (live foetuses of dams surviving until scheduled necropsy on Day 21 post-coitum).
- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: No
Live foetuses will be euthanised by oral administration of sodium pentobarbital. In case this is not possible due to a malformation, the foetus will be euthanised by interscapular injection of sodium pentobarbital.
Malformed foetuses will be collected and fixed in the most appropriate fixative (based on type of malformation).
Malformed late resorptions will be collected and fixed in 10 % buffered formalin.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: Most animals presented with erected fur, hunched posture and decreased activity from Days 6 - 8 post-coitum onwards, together with pallor skin from Day 15 - 17 post-coitum onwards. Furthermore, abnormal breathing sounds (3/6 animals) and weak appearance (2/6 animals) were noted on intermittent days between Days 16 - 20 post-coitum. Additionally, salivation was noted for all animals on intermittent days between Day 10 - 19 post-coitum.

50 mg/kg/day: All animals presented with erected fur, hunched posture and decreased activity from Day 6 - 7 post-coitum onwards. Furthermore, abnormal breathing sounds (2/6 animals), pallor skin (3/6 animals) and weak appearance (1/6 animals) were noted on intermittent days between Days 12 - 15 post-coitum. Additionally, salivation was noted for all animals from Day 8 post-coitum onwards.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

25 mg/kg/day: No mortality occurred during the study period.
50 mg/kg/day: All animals were sacrificed on Day 15 post-coitum; 4/6 animals (Nos. 8, 9, 11, 12) were sacrificed for humane reasons, because of severe body weight loss (10 - 15 % between Days 12 - 15 post-coitum), together with low food consumption. As at that point sufficient data was available to conclude that 50 mg/kg/day was too high to tolerate, the remaining two animals at 50 mg/kg/day (Nos. 7 and 10) were sacrificed as well.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: Absent (2/6 animals) or low (4/6 animals) body weight gain was noted between Days 6 - 9 post-coitum. A slight recovery was noted for 5/6 animals from Day 9 post-coitum onwards, whereas body weight loss was noted for 1/6 animals (No. 3) from Day 12 post-coitum until the end of the treatment period (up to 12 % at Day 21 post-coitum, compared to Day 12 post-coitum). Additionally, body weight loss (5 %) was noted for Animal No. 5 between Days 18 - 21 post-coitum. At Day 21 post-coitum, body weights of these 2/6 animals were below the lower limit of the historical control range. Normal body weight was noted for the remaining 4/6 animals. Mean gravid uterus weight was considered within normal range. After correction for gravid uterus weight, 3/6 animals had body weight loss (up to severe), 1/6 animals had low body weight gain and 2/6 had normal body weight gain.
50 mg/kg/day: Low body weight gain (5/6) or body weight loss (1/6) (3 % compared to Day 6 post-coitum) were noted between Day 6 - 9 post-coitum. A slight recovery was noted for 5/6 animals between Days 9 - 12 post-coitum, followed by low body weight gain (2/6 animals) or marked body weight loss (4/6 animals) between Day 12 - 15 post-coitum.

Historical Control Data:
Body weights (gram) of time-mated F0-females (period 2015 – Week 20 2020) at:
Day 2 – mean = 207; P5 – P95 = 180 - 237 (n = 1 248)
Day 6 – mean = 223; P5 – P95 = 194 - 254 (n = 1 296)
Day 9 – mean = 232; P5 – P95 = 203 - 264 (n = 1 296)
Day 12 – mean = 247; P5 – P95 = 203 - 264 (n = 1 296)
Day 15 – mean = 261; P5 – P95 = 229 - 297 (n = 1 296)
Day 18 – mean = 291; P5 – P95 = 253 - 331 (n = 1 296)
Day 21 – mean = 326; P5 – P95 = 280 - 374 (n = 1 296)

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: Mean absolute liver weights were considered within normal range. Mean relative liver weights were at the high end of the historical control range but were considered secondary to the decreased terminal body weights. Mean absolute and relative thymus weights were below the lower limit of the historical control range.
50 mg/kg/day: Organ weights were not collected as animals did not survive until scheduled necropsy.


Historical Control Data:
Gravid uterine weight and corrected body weights of time-mated F0-females (period 2015 – Week 42 2019):
Gravid Uterus Weight (gram) – mean = 75; P5 – P95 = 48 - 96 (n = 1311)
Adjusted BWG (gram) – mean = 29; P5 – P95 = 15 - 44 (n = 1311)

Organ weights of time-mated F0-females (gram; period 2016 – Week 20 2020):
Liver – mean = 11.42; P5 – P95 = 8.73 - 15.09 (n = 226)
Thymus – mean = 0.347; P5 – P95 = 0.224 - 0.513 (n = 22)

Organ weights relative to body weights of time-mated F0-females (%; period 2016 – Week 20 2020):
Liver – mean = 3.55; P5 – P95 = 2.91 - 4.49 (n = 226)
Thymus – mean = 0.107; P5 – P95 = 0.068 - 0.153 (n = 22)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: A small thymus was noted in 3/6 animals (in line with the low thymus weights).
50 mg/kg/day: No macroscopic findings were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Food consumption
25 mg/kg/day: Food consumption was considered low for all animals from Day 6 post-coitum onwards. Food consumption was notably more affected in 2/6 females that showed body weight loss during the treatment period.
50 mg/kg/day: Food consumption was considered low for all animals from Day 6 post-coitum onwards.

Historical Control Data:
Food consumption (gram/animal/day) of time-mated F0-females (period 2015 – Week 20 2020) at:
Days 2 - 6 – mean = 21; P5 – P95 = 16 - 26 (n = 1270)
Days 6 - 9 – mean = 20; P5 – P95 = 15 - 25 (n = 1294)
Days 9 - 12 – mean = 21; P5 – P95 = 16 - 26 (n = 1296)
Days 12 - 15 – mean = 22; P5 – P95 = 17 - 27 (n = 1293)
Days 15 - 18 – mean = 23; P5 – P95 = 18 - 28 (n = 1296)
Days 18 - 21 – mean = 23; P5 – P95 = 18 - 28 (n = 1289)

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

25 mg/kg/day: The mean number of pre- and post-implantation loss were considered within normal range.

Historical Control Data:
Number of Implantations – mean = 11.2; min – max = 10.0 - 12.5 (n = 1 321 dams)
Pre-implantation loss (%) – mean = 6.5; min – max = 0.8 - 14.6 (n = 1 007 dams)
Post-implantation Loss (%) – mean = 5.2; min – max = 1.5 - 11.6 (n = 1 293 dams with live foetuses)

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

25 mg/kg/day: The mean number of early and late resorptions were considered within normal range.
50 mg/kg/day: The mean number of early resorptions were considered within normal range.

Dead fetuses:

no effects observed

Description (incidence and severity):

25 mg/kg/day: The mean number of live and dead foetuses were considered within normal range.
50 mg/kg/day: No foetal data was collected as animals did not survive until scheduled necropsy.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

25 mg/kg/day: All females were pregnant with live foetuses at scheduled necropsy (Day 21 post-coitum).
50 mg/kg/day: All animals were pregnant with live embryos at the day of sacrifice (Day 15 post-coitum).

Historical Control Data:
Maternal pregnancy of time-mated F0-females (period 2016 – 2020):
Pregnant females (%) – mean = 98.3; min – max = 90.9 - 100 (n = 1 321 dams)

Other effects:

no effects observed

Description (incidence and severity):

25 mg/kg/day: The mean number of corpora lutea and implantation sites were considered within normal range.
50 mg/kg/day: The mean number of corpora lutea and implantation sites were considered within normal range.

Historical Control Data:
Number of Corpora Lutea – mean = 12.0; min – max = 11.1 - 13.7 (n = 1 321 dams)

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Remarks:

This is a dose range-finding study and was not designed to determine the NOAEL.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: Mean male, female and total foetal body weights were below the lower limit of the Historical Control Data.
50 mg/kg/day: No foetal data was collected as animals did not survive until scheduled necropsy.

Historical Control Data:
Mean Foetal Weight Males (gram) – mean = 5.4; min – max = 5.0 - 5.5 (n = 6 774 foetuses)
Mean Foetal Weight Females (gram) – mean = 5.1; min – max =4.7 - 5.3 (n = 7 024 foetuses)
Mean Foetal Weight All (gram) – mean = 5.3; min – max = 5.0 - 5.4 (n = 13 798 foetuses)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

25 mg/kg/day: The mean number of live and dead foetuses were considered within normal range.
50 mg/kg/day: No foetal data was collected as animals did not survive until scheduled necropsy.

Historical Control Data:
Number of Live Foetuses – mean = 10.7; min – max = 9.5 - 12.1 (n = 1 293 dams with live foetuses)

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: The mean percentage of Live Male and Female Foetuses per Litter showed a trend towards an increased ratio of Male over Female Foetuses, mainly contributed to Litter Nos. 2 and 3.
50 mg/kg/day: No foetal data was collected as animals did not survive until scheduled necropsy.

Historical Control Data:
Live Male Foetus/Litter (%) – mean = 48.9; min – max = 40.6 - 56.5 (n = 6 774 foetuses)
Live Female Foetus/Litter (%) – mean = 51.1; min – max = 43.5 - 59.4 (n = 7 024 foetuses)

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

25 mg/kg/day: No external foetal abnormalities were noted.
50 mg/kg/day: No foetal data was collected as animals did not survive until scheduled necropsy.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg/day: Mean placental weight for male and female foetuses was below the lower limit of the Historical Control Data.
50 mg/kg/day: No foetal data was collected as animals did not survive until scheduled necropsy.

Historical Control Data:
Live Male Foetuses Pl. Wt (gram) – mean = 0.51; min – max = 0.48 - 0.57 (n = 6 774 foetuses)
Live Female Foetuses Pl. Wt (gram) – mean = 0.49; min – max = 0.47 - 0.55 (n = 7 024 foetuses)

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Remarks:

This is a dose range-finding study and was not designed to determine the NOAEL.

Abnormalities:

not examined

Developmental effects observed:

not specified

Summary of Clinical Observations: Gestation

Observation Type	25 mg/kg/day Group	50 mg/kg/day Group
Salivation:
Number of Animals Affected	6	5
Number of Times Recorded	16	14
% of Affected Animals	100	83
First to Last Seen	10 - 19	8 - 14
Breathing, Abnormal Sounds:
Number of Animals Affected	3	2
Number of Times Recorded	4	3
% of Affected Animals	50	33
First to Last Seen	16 - 20	12 - 14
Hunched Posture:
Number of Animals Affected	5	6
Number of Times Recorded	14	23
% of Affected Animals	83	100
First to Last Seen	7 - 21	7 - 15
Fur, Erected:
Number of Animals Affected	6	6
Number of Times Recorded	88	57
% of Affected Animals	100	100
First to Last Seen	6 - 21	6 - 15
Skin, Pallor:
Number of Animals Affected	6	3
Number of Times Recorded	37	3
% of Affected Animals	100	50
First to Last Seen	15 - 21	15 - 15
Weak
Number of Animals Affected	2	1
Number of Times Recorded	3	1
% of Affected Animals	33	17
First to Last Seen	16 – 20	15 - 15
Activity Decreased:
Number of Animals Affected	5	6
Number of Times Recorded	29	26
% of Affected Animals	83	100
First to Last Seen	8 - 21	7 - 14

 

Summary of Body Weights: Gestation

Group		Day(s) Relative to Mating (Litter A)
		2	6	9	12	15	18	21
25 mg/kg/day – Group 1	Mean	211.2	232.2	235.3	247.7	255.0	279.2	296.3
	SD	13.9	15.7	13.7	16.4	21.6	34.1	49.1
	N	6	6	6	6	6	6	6
50 mg/kg/day – Group 1	Mean	205.8	220.8	224.0	233.3	215.3	-	-
	SD	7.3	9.5	11.5	8.6	20.6	-	-
	N	6	6	6	6	6	-	-

 

Summary of Macroscopic Pathology: Terminal Euthanasia

Group	25 mg/kg/day
Number of animals	6
Pancreas
Submitted	1
Abnormal appearance; gelatinous	1
Thymus
Submitted	3
Small	3
Whole animal
No visible lesions	3

 

Summary of Macroscopic Pathology: Unscheduled Euthanasia

Group	50 mg/kg/day
Number of animals	6
Whole animal
No visible lesions	6

 

Summary of Maternal Performance and Mortality

Parameter		25 mg/kg/day Group 1	50 mg/kg/day Group 2
Group size		6	6
Number of females pregnant	N+ve	6	6
	%	100	100
Female with live foetuses	N+ve	6	6
	%	100	100
Total resorptions	N+ve	0	0
	%	0	0
Female with all nonviable	N+ve	0	0
	%	0	0
Terminal euthanasia	N+ve	6	0
	%	100	0
Unscheduled death/ euthanasia	N+ve	0	6
	%	0	100
Unscheduled euthanasia	N+ve	0	6
	%	0	100

 

Summary of Ovarian and Uterine Examinations and Litter Observations

Parameter		25 mg/kg/day Group 1
Female with live foetuses	N+ve	6
	%	100
Number of corpora lutea	Mean	12.5
	SD	1
	N	6
Number of implantations	Mean	12
	SD	1.4
	N	6
Pre-implantation loss (%)	Mean	4.06
	SD	6.90
	N	6
Total number of foetuses	Mean	12
	SD	1.4
	N	6
Number of live foetuses	Mean	12
	SD	1.4
	N	6
Number of dead foetuses	Mean	0
	SD	0
	N	6
Number of early resorptions	Mean	0
	SD	0
	N	6
Number of late resorptions	Mean	0
	SD	0
	N	6
Total number of resorptions	Mean	0
	SD	0
	N	6
Post-implantation loss (%)	Mean	0
	SD	0
	N	6

Summary of Ovarian and Uterine Examinations and Litter Observations

Parameter		25 mg/kg/day Group 1
Number of live male foetuses	Mean	7.5
	SD	2.2
	N	6
Number of live female foetuses	Mean	4.5
	SD	1.6
	N	6
Live male foetus/litter (%)	Mean	62.09
	SD	14.39
	N	6
Live female foetus/litter (%)	Mean	37.91
	SD	14.39
	N	6
Mean foetal weight males (g)	Mean	4.590
	SD	0.858
	N	6
Mean foetal weight females (g)	Mean	4.433
	SD	0.877
	N	6
Mean foetal weight all (g)	Mean	0.453
	SD	0.868
	N	6
Live male foetuses pl. wt (mean)	Mean	0.453
	SD	0.056
	N	6
Live female foetuses pl. wt (mean)	Mean	0.427
	SD	0.077
	N	6
Live mean placental weight (g)	Mean	0.440
	SD	0.061
	N	6

Conclusions:

Under the conditions of this study, selected dose levels for the subsequent Prenatal Developmental Toxicity study by oral gavage are 3, 10 or 25 mg/kg/day, on the basis that a dose of 50 mg/kg/day was clearly in excess of the maximum tolerated dose being associated with severe body weight loss and low food consumption, necessitating the early termination of the group. The dose level of 25 mg/kg bw/day was associated with clear but tolerable maternal toxicity in terms of clinical signs, reduced body weight and body weight gain, lower food intake, lower absolute and relative thymus weight with macroscopic correlate. Therefore it was considered that a dose of 25 mg/kg/day would adequately demonstrate systemic toxicity in accordance with the OECD 414 test guideline requirements. Dose levels of 3 and 10 mg/kg/day were selected on the expectation they would characterise a dose-response, with the lowest dose expected to be a No Adverse Effect Level (NOAEL).

Executive summary:

The dose range-finding study was conducted to determine the highest tolerated dose level of the test material orally by gavage in pregnant rats. This study was performed as a Dose Range Finding study for the subsequent Prenatal Developmental Toxicity study by oral gavage and as such was not conducted to a guideline or GLP.

Timed-pregnant Wistar-Han rats were administered the test material at a dose of either 25 or 50 mg/kg/day by oral gavage from Day 6 up to and including Day 20 post-coitum.  At intervals throughout the day, mortality and cage side clinical observations were recorded. Every third day during treatment and on the day of necropsy, detailed clinical observations, body weights, food consumption were recorded.  Following termination on Day 21 (group 1) or Day 15 post-coitum (early termination) external, thoracic and abdominal examinations were made, with special attention being paid to the reproductive organs. Number of corpora lutea, weight of the liver, thymus and uterus, placental weights of live foetuses, number of implantation sites, number and distribution of live and dead foetuses, number and distribution of early and late resorptions were determined. External examination, external sex determination and body weight were made of live foetuses of dams surviving until scheduled necropsy on Day 21 post-coitum.

A dose of 50 mg/kg/day was clearly in excess of the maximum tolerated dose, being associated with severe body weight loss and low food consumption, necessitating the early termination of the group. The dose level of 25 mg/kg bw/day was associated with clear but tolerable maternal toxicity in terms of clinical signs, reduced body weight and body weight gain, lower food intake, lower absolute and relative thymus weight with macroscopic correlate. Therefore, it was considered that a dose of 25 mg/kg/day would adequately demonstrate systemic toxicity in accordance with the OECD 414 test guideline requirements. Dose levels of 3 and 10 mg/kg/day were selected on the expectation they would characterise a dose-response, with the lowest dose expected to be a No Adverse Effect Level (NOAEL).

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2021 to 30 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Adjustment will be made for specific gravity of the vehicle and test material. No correction will be made for the purity/composition of the test material.
Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and were not used for dosing.

Species:

rat

Strain:

Wistar

Remarks:

Wistar-Han, Outbred, SPF-Quality.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 - 14 weeks old.
- Weight at study initiation: 186 – 266 g.
- Housing: Animals will be individually housed in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilised wooden fibers as bedding material.
- Diet: Ad libitum, except during designated procedures.
- Water: Freely available to each animal via water bottles.
- Acclimation period: Five days.

ENVIRONMENTAL CONDITIONS
- Temperature: The actual daily mean temperature during the study period was 22 °C.
- Humidity: The actual daily mean relative humidity was 44 to 49 %.
- Air changes: At least 10 air changes per hour.
- Photoperiod: 12-hours light and 12-hours dark (may be interrupted for designated procedures).

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Stability for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator is confirmed over the concentration range 1 to 250 mg/mL (suspensions).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose volume: 5 mL/kg.
- Dose levels were based on the results of the dose range finding study.
- Dose concentration:
0 mg/kg/day: 0 mg/mL
5 mg/kg/day: 1 mg/mL
10 mg/kg/day: 2 mg/mL
25 mg/kg/day: 5 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SUMMARY
The objective of the analytical study was to determine the accuracy of preparation and homogeneity of 2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4- stannatetradecanoate (DMTE) in formulations.
Week 1: preparation date 03 May 2021
A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the Group 2 samples was 0.50 %.
The concentrations analysed in the formulations of Groups 2, 3 and 4 were slightly below the target range of 85 – 115 % (i.e. mean accuracies 83 %, 82 % and 83 % respectively). It was decided to re-analyse the samples and at the same time to perform additional analysis of formulations that were freshly prepared on 04 May 2021.
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Week 1: re-analysis Groups 2, 3 and 4 preparation date 03 May 2021
The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %).
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Week 1: preparation date 04 May 2021
A significant response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the Group 2 samples was 29 %.
New Group 1 samples were taken for analysis from the same formulation. In addition, a new formulation was prepared for Group 1 sampling and analysis on 07 May 2021.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %).
Both formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Week 1: re-analysis Group 1 preparation date 04 May 2021
No test material was detected in the formulation.
Week 1: Group 1 preparation date 07 May 2021
No test material was detected in the formulation

METHOD
Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector :Acquity UPLC TUV or PDA detector (Waters)
Column: Acquity UPLC BEH C18, 50 mm  2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature: 40 °C ± 1 °C
Injection volume: 50 µL
Solvent A: Acetonitrile
Solvent B: 0.1 % formic acid in water
Gradient:
0.0 min: 50 % solvent A, 50 % solvent B
0.5 min: 50 % solvent A, 50 % solvent B
1.5 min: 80 % solvent A, 20 % solvent B
3.0 min: 80 % solvent A, 20 % solvent B
3.1 min: 50 % solvent A, 50 % solvent B
4.5 min: 50 % solvent A, 50 % solvent B
Flow: 0.6 mL/min
UV detection: 210 nm

Study Samples
Accuracy and homogeneity were determined for formulations prepared for use in Week 1.
The chemical analyses were conducted as specified below. Samples and remaining formulation were stored in normal glassware causing the samples stored at room temperature to be exposed to normal laboratory light conditions.
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10 or 25 mL. For determination of accuracy, samples were taken at middle position (50 % height) or at top, middle and bottom position (90 %, 50 % and 10 % height). The samples taken at 90 %, 50 % and 10 % height were also used for the determination of the homogeneity of the formulations.
The volumetric flasks were filled up to the mark with acetonitrile and left to stand for 30 minutes. The solutions were further diluted to obtain an end solution of 50/50 (v/v) acetonitrile/water and concentrations within the calibration range.
Preparation of Solutions Stock and Spiking Solutions:
- Stock and spiking solutions of the test material were prepared in acetonitrile at concentrations of 2000 and 5000 mg/L.
- Calibration Solutions: Six calibration solutions in the concentration range of 0.8 - 40 mg/L were prepared in from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.
- Quality Control (QC) Samples: Approximately 500 mg blank vehicle was spiked with the test material at a target concentration of 1 or 5 mg/mL. The QC samples were treated similarly as the study samples.
Sample Injections
- Calibration solutions were injected in duplicate. Study samples and QC samples were analysed by single injection.

Constructed Variables
Response (R) [units]: Peak area of the test material
Calibration curve:
R = a CN + b
Where:
CN: Nominal concentration [mg/L]
a: Slope [units  L/mg]
b: Intercept [units]

Volume sample for accuracy samples [mL]:
V = ((Wveh / ρveh) + (Wti / ρti))

Where:
Wve: Weight vehicle [g]
Wti: Weight test material [g]
ρveh: Density of vehicle [g/mL]
ρti: Density of test material [g/mL]

Volume sample (v) for formulation samples [mL]:

V = w / ρ

Where:
W = Weight formulation sample (g)
ρ = Density of formulation (g / mL)

Analysed concentration (CA) [mg/mL]:

CA = ((R - b) / a) × ((V x d x 10^-3) / v)

Where:
V: volume volumetric flask [mL]
d: Dilution factor
v: Volume accuracy sample or formulation sample [mL]

Accuracy of QC samples [%]:

CA / CN × 100

Where:
CN: nominal concentration [mg/g]

Accuracy of study samples [%]
CA / CT × 100

Where:
CT: Target concentration [mg/g]
Maximum contribution:

(𝑅𝑏𝑙𝑎𝑛𝑐𝑜 ×𝑑 𝑏𝑙𝑎𝑛𝑐𝑜) / (𝑅𝑠𝑎𝑚𝑝𝑙𝑒 × 𝑑𝑠𝑎𝑚𝑝𝑙𝑒) . 100 %

Where:
R: Response
d: Dilution factor

Formulations were suspensions. Concentration results were considered acceptable if mean sample concentration results were within or equal to 85 - 115 % of target concentration. Homogeneity results were considered acceptable if the coefficient of variation was ≤ 10 %.

RESULTS
Density of each formulation measured on 28 Apr 2021 using a DMA 35 portable density meter (Anton Paar, Graz, Austria) is:
Group 2: 0.8849 g/mL
Group 3: 0.8851 g/mL
Group 4: 0.8860 g/mL

Calibration Curves
Calibration curves were constructed using six concentration levels. For each level, duplicate responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration^2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

QC Samples
The mean accuracies of the QC samples analysed on 03 May 2021 and 05 May 2021 were within the criterion range of 85 – 115 %. It demonstrated that the analytical method was adequate for the determination of the test material in the study samples. The mean accuracy of the QC samples analysed on 07 May 2021 was below the criterion range of 85 – 115 % (i.e. 79 %). However, purpose of analysis performed on 07 May 2021 was to demonstrate absence of the test material in the Group 1 formulation, thus lower accuracy of the QC samples does not play a significant role in this analysis.
Study Samples
Week 1: preparation date 03 May 2021
A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the Group 2 samples was 0.50 %.
The concentrations analysed in the formulations of Group 2, 3 and 4 were below the target range of 85-115 % (i.e. mean accuracies 83 %, 82 % and 83 % respectively). An out of specification investigation has been performed in which equipment, samples, analytical method, sample treatment and the raw data were reviewed. Conclusion of the investigation was that the extraction of the samples from the vehicles may not have been efficient which had an effect of the recovery of the samples. It was decided to re-analyse the samples and at the same time perform additional analysis of the formulations that were freshly prepared on 04 May 2021.
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).

Week 1: re-analysis Group 2, 3 and 4 preparation date 03 May 2021
The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %).
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).

Week 1: preparation date 04 May 2021
A significant response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the Group 2 samples was 29 %. An out of specification investigation has been performed in which equipment, samples, analytical method, sample treatment and the raw data were reviewed. Conclusion of the investigation was that that it was caused by contamination of Group 1 samples after sampling. To confirm this, new Group 1 samples were taken from the same formulation. In addition, a new formulation was prepared for Group 1 sampling and analysis on 07 May 2021.
The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %).
Both formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).

Week 1: re-analysis Group 1 preparation date 04 May 2021
No test material was detected in the formulation. The finding confirms that contamination of the Group 1 samples after sampling was the cause of their high response obtained during analysis on 05 May 2021.
No test material was detected in the Group 1 formulation prepared on 07 May 2021.

Week 1: Group 1 preparation date 07 May 2021
No test material was detected in the formulation.


Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records.

Dose Formulation Analyses
Group 2, 3 and 4 concentrations were slightly below target on 03 May 2021. These samples, and freshly prepared samples were reanalysed on 04 May 2021. In the freshly prepared samples, test material was detected in Group 1. These samples and freshly prepared samples were then reanalysed on 07 May 2021.
Week 1: preparation date 03 May 2021
A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the Group 2 samples was 0.50 %. This response was considered negligible and considered to have no impact on the outcome of the study.
The concentrations analysed in the formulations of Groups 2, 3 and 4 were slightly below the target range of 85 – 115 % (i.e. mean accuracies 82 %, 83 % and 82 % respectively). Conclusion of the out of specification investigation was that the extraction of the samples from the vehicles may not have been efficient which had an effect of the recovery of the samples. It was decided to re-analyse the samples and at the same time to perform additional analysis of formulations that were freshly prepared on 04 May 2021.
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Week 1: re-analysis Groups 2, 3 and 4 preparation date 03 May 2021
The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %).
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Week 1: preparation date 04 May 2021
A significant response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. The maximum contribution to the Group 2 samples was 29 %. Conclusion of the out of specification investigation was that that Group 1 samples were contaminated after sampling.
New Group 1 samples were taken for analysis from the same formulation. In addition, a new formulation was prepared for Group 1 sampling and analysis on 07 May 2021.
The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85 % and 115 %).
Both formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Week 1: re-analysis Group 1 preparation date 04 May 2021
No test material was detected in the formulation.
Week 1: Group 1 preparation date 07 May 2021
No test material was detected in the formulation.
In summary: As re-analysis of Groups 2, 3, and 4 samples were in agreement with target concentrations and re-analysis of Group 1 samples were without detection of test material, it can be concluded that animals received accurate doses. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Details on mating procedure:

- Impregnation procedure: Timed pregnant. Untreated females will be mated at the Supplier and will be at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum.

Frequency of treatment:

Once daily

Duration of test:

Day 6 to Day 21 post-coitum.

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.
The dose levels were selected based on the results of a Dose Range Finding Study with the test material in consultation with the Study Monitor and in an attempt to produce graded responses to the test material.
Based on the results of the dose range finding study (2 groups, 25 and 50 mg/kg/day), a dose level of 25 mg/kg/day was selected to be the top dose level for the current main study.
The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, all parental animals.
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy. 1 hour post-dose.
- Cage side observations: Animals will be observed within their cage unless necessary for identification or confirmation of possible findings. Cage debris will be examined to detect premature birth, if applicable.
Mortality: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes, all parental animals.
- Time schedule: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. Animals are removed from the cage.

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

FOOD CONSUMPTION: Yes quantitatively measured for all parental animals.
- Time schedule: Over Days 2 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.

WATER CONSUMPTION: Yes, all parental animals.
- Time schedule for examinations: Regular basis throughout the study. Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.


POST-MORTEM EXAMINATIONS: Yes. Animals surviving until scheduled euthanasia on Day 21 post-coitum will be euthanised using the carbon dioxide inhalation (gradual fill) procedure and subsequently exsanguinated. No body weight will be recorded at necropsy.
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) will be subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities will be recorded, collected and fixed in the appropriate fixation, together with the animal identification; collection of specific macroscopic abnormalities may be omitted only at the discretion of the Study Director.
- Organ Weights – F0-Generation: The liver, thymus, thyroid gland and uterus will be weighed at necropsy for all scheduled euthanasia animals, except for females that delivered their offspring early. Organ weights will also not be recorded for animals found dead, euthanised in poor condition or in extremis or that delivered their offspring early. Paired organs will be weighed together. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) will be calculated.
- Tissue Collection and Preservation – F0-Generation: The liver, thymus, thyroid gland and macroscopic abnormalities will be collected from all animals and preserved in 10 % buffered formalin.
- Histology – F0-Generation: Thyroid gland of all animals will be embedded in paraffin, sectioned at a thickness of 2 - 4 micrometres, mounted on glass slides, and stained with hematoxylin and eosin.


OTHER:
CLINICAL PATHOLOGY
All F0-animals, Day 21 post-coitum. Non-fasted. 1.0 mL sampled between 07.00 and 09.00 from the jugular vein. The order of groups will be randomised per collection day.
Thyroid hormone: After receipt of the serum for T3, T4 and TSH analysis it will be divided in two aliquots. One aliquot will be used for measurement of thyroid hormones using the IMMULITE® 1000 analyser (TSH). These samples will be stored in an ultra-low freezer (≤ -75 °C) until analysis. The other aliquot will be used for measurement of T3 and T4 using LC-MS. The aliquots for
T3 and T4 will be collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and stored in a freezer (≤ -15 °C) until analysis. Measurement will be performed according to the bioanalytical method validated in the Test Facility.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. Each ovary and uterine horn of all animals will be dissected and examined as quickly as possible as part of the necropsy procedure. For animals found dead, sacrificed before planned necropsy or that started to deliver, these findings will be reported in the individual data tables only.
Examinations included:
- Gravid uterus weight: Yes. Not assessed for animals found dead, sacrificed before planned necropsy or that started to deliver.
- Number of corpora lutea: Yes
- Number of implantations: Yes. In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique in order to detect any former implantation sites.
- Number of early resorptions: Yes, the number and distribution.
- Number of late resorptions: Yes, the number and distribution.
Other:
- The number and distribution of live and dead foetuses.
- Placental weights of live foetuses only (after weighing, placentae will not be retained).

Fetal examinations:

Method of Euthanasia and Foetal External Examinations – F1-Generation:
Live foetuses will be euthanised by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. In case this is not possible due to a malformation, the foetus will be euthanised by decapitation or by interscapular injection of sodium pentobarbital. Recognizable live foetuses of females found dead or sacrificed before planned necropsy or pups from females that delivered early, will be euthanised by decapitation.
Malformed late resorptions will be collected and fixed in 10 % buffered formalin. Late resorptions without malformations will be discarded.

- External examinations: Yes: The following will have external examinations conducted:
Viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.
Non-viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.
Late resorption.
Foetus of dam not surviving until Day 21 post-coitum. Dams that are found dead, sacrificed before planned necropsy or that started to deliver.

- Soft tissue examinations: Yes: Viable and non-viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum. 100 % will have internal sex confirmation. Visceral body examinations will be conducted on around 50 % of viable and non viable foetuses of dams surviving until scheduled necropsy on Day 21 post-coitum. Fresh (non-fixed state) abnormalities may be collected and fixed at the discretion of the Study Director. Foetuses will be eviscerated, fixed in 96 % aqueous ethanol, macerated in potassium hydroxide and processed for double staining (Alcian Blue 8GX and Alizarin Red S) but not examined in first instance.
- Skeletal examinations: Yes: Around 50 % of foetuses with head. Foetuses will be eviscerated, fixed in 96 % aqueous ethanol, macerated in potassium hydroxide and processed for double staining (Alcian Blue 8GX and Alizarin Red S). Specimens will be archived in glycerin with bronopol as preservative.
- Head examinations: Yes: Around 50 % of foetuses viscerally screened. Fixed in Bouin’s solution, tissues without variations or malformations will be discarded. Tissues with variations or malformations will be stored.
- Anogenital distance of all live rodent pups: The sex of each foetus based on the anogenital distance, if possible (not for animals found dead, sacrificed before planned necropsy or that started to deliver). Anogenital distance will be measured for viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.
The following will have external sex determination conducted:
Viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.
Non-viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.
- The following will have body weight examinations conducted:
Viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.
Non-viable foetus of dam surviving until scheduled necropsy on Day 21 post-coitum.

External, visceral, and skeletal findings will be recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
In the case of (suspected) visceral malformations in foetuses that are selected for skeletal examination, this foetus will only be examined for visceral abnormalities in first instance.
In the case of (suspected) skeletal malformations in foetuses that are selected for visceral examination, this foetus will only be examined for skeletal abnormalities in first instance.

Statistics:

See below.

Indices:

Pregnancy Rate (%): (No. of pregnant females/ No. of mated females) x 100
Male Foetuses (%): (No. of male foetuses / No. of foetuses) x 100
Female Foetuses (%): (No. of female foetuses / No. of foetuses) x 100
Pre-Implantation Loss (%): ((No. of corpora lutea – No. of implantations) / No. of corpora lutea) x 100
Post-Implantation Loss (%): ((No. of implantations – No. of live foetuses) / No. of implantations) x 100
Litter % of Foetuses with Abnormalities: (No. of foetuses in litter with a given finding / No. of foetuses in litter examined) x 100

Historical control data:

Study date range: 2016 – 2020
Number of studies: 60
Total number of foetuses / litters examined externally: 13 798 / 1 293
Total number of foetuses / litters examined viscerally: 6 911 / 1 292
Total number of foetuses / litters examined skeletally: 6 905 / 1 291

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 25 mg/kg/day, hunched posture was noted for eight females between Days 10 - 13 post-coitum. This was considered related to treatment with the test material.
At 10 mg/kg/day, hunched posture was noted for a single female between Days 11 - 12 post-coitum. Given the low incidence and the short duration, this was considered not toxicologically relevant. Erected fur, decreased activity and abnormal breathing were noted in females of all group. As these signs were also noted in the control group, they were considered not toxicologically relevant. Salivation seen after dosing in all groups, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response to the taste of the dose formulation rather than a sign of systemic toxicity. Incidental findings (head tilt, fur loss, thin fur, skin scab) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered related to treatment with the test material.
Of note: Female No. 18 (control group) delivered its litter early on the day of scheduled necropsy and Female No. 51 (10 mg/kg/day) delivered its litter after Terminal Body Weight measurement and blood withdrawal on the day of scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 25 mg/kg/day, mean body weights and body weight gain of treated females remained in the same range as control up to Day 18 post-coitum. Between Days 18 - 21 post-coitum, mean body weight gain was lower than control (not statistically significant), resulting in a lower mean body weight at Day 21 post-coitum (2.4 % lower than control; not statistically significant). As this decrease was noted at the end of the treatment period, this was considered toxicologically relevant and related to treatment with the test material.
Mean body weights and body weight gain at 10 and 5 mg/kg/day remained in the same range as control over the treatment period.
Mean gravid uterus weight of treated females remained in the same range as control.
Mean body weight gain adjusted for gravid uterus was reduced at 25 mg/kg/day (68 % lower than control) and at 10 mg/kg/day (31 % lower than control; not statistically significant). Moreover, mean values of these groups were below the lower end of the available Historical Control Data.
Mean body weight gain corrected for gravid uterus at 5 mg/kg/day was considered to be in the same range as control.

Historical control data for pregnant Wistar Han rats (period 2015 - 2020)
Corrected body weight gain (g) mean: 29.0, P5 - P95: 14.9 – 43.4 (n = 1278)

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Of Note: Female No. 40 (5 mg/kg/day) was non-gravid. Female Nos. 18 (control group) and 51 (10 mg/kg/day) delivered their litter on the day of scheduled necropsy.
There were no test material-related alterations in thyroid gland weights. Absolute and relative thymus weights at 25 mg/kg/day were lower than control (17 % and 15 %, respectively; not statistically significant for relative thymus weights).
Absolute and relative thymus weights at 10 and 5 mg/kg/day were considered to be similar to control.
Absolute and relative liver weights of all treated groups were considered to be similar to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related gross observations in the thyroid glands.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related microscopic observations in the thyroid glands.
All the recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Description (incidence and severity):

Food consumption:
At 25 mg/kg/day, mean food consumption was decreased (9 % lower than control) when considering the total treatment period (Days 6 to 21 post-coitum).
Mean food consumption at 10 and 5 mg/kg/day were similar to control.

Thyroid Hormone Analyses
Of Note: Female No. 40 (5 mg/kg/day) was non-gravid. Female No. 18 (control group) delivered its litter early on the day of scheduled necropsy. These females were excluded from the summary data tables. Female No. 51 (10 mg/kg/day) delivered its litter after blood withdrawal and was therefore not excluded. For Female No. 2 (control group), T3 and T4 levels could not be measured due to insufficient amounts of serum.
Slightly increased serum levels of TSH were noted at 25 mg/kg/day (1.24x of control) and slightly decreased serum levels of TSH were noted at 5 mg/kg/day (0.77x of control), however without reaching statistical significance. As mean levels remained within the available historical control data and no dose response was noted, these slight differences in TSH were therefore considered not to be test material-related.
Serum levels of total triiodothyronine (T3) and total thyroxine (T4) were considered to be unaffected by treatment with the test item up to 25 mg/kg/day.
Historical control data for pregnant Wistar Han rats (period 2016 - 2020): TSH (mU/L) mean: 0.353, P5 - P95: 0.127– 0.699 (n = 373)

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean numbers of implantation sites and post-implantation loss in the control and test groups were similar and in the range of normal biological variation.
At 5 and 25 mg/kg/day, the pre-implantation loss was slightly increased (9.3 % and 9.1 %, respectively vs. 7.0 % in control, not statistically significant). This was mostly due to Female Nos. 29 (5 mg/kg/day) and 88 (25 mg/kg/day) with low number of implantations (5 and 4, respectively) and excluding their values, the mean pre-implantation loss was 6.9 % and 6.5 %. As the treatment with test material is started after implantation (Day 6 post-coitum), and in the absence of a clear dose-dependent relation, this was considered not related to treatment with the test material.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean numbers of early and late resorptions in the control and test groups were similar and in the range of normal biological variation.

Dead fetuses:

not specified

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not specified

Description (incidence and severity):

At scheduled necropsy, Female No. 40 (5 mg/kg/day) was non-gravid. Female Nos. 18 (control group) and 51 (10 mg/kg/day) delivered their litter on the day of scheduled necropsy.
The number of females with viable litters for evaluation was 21, 21, 21 and 22 in the control, 5, 10 and 25 mg/kg/day groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea in the control and test groups were similar and in the range of normal biological variation.
There were no test material-related effects on placental weights (male, female and combined) noted after treatment with the test material up to 25 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: Food consumption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal body weights for all treated groups were higher than the control group, reaching statistical significance at 5 mg/kg/day for male, female and combined foetal body weight. However, mean foetal body weights (male, female and combined) for all treated groups were within normal range of the available Historical Control Data and in the absence of a dose-dependent response, these results were considered unaffected by treatment with the test material.
Historical control data for body weight of Wistar Han foetuses (period 2016 - 2020):
Males (g): mean: 5.4; P5 - P95: 5.2 – 5.5 (n= 6 774)
Females (g): mean: 5.1; P5 - P95: 4.9 – 5.3 (n= 7 024)
Combined (g): mean: 5.3; P5 - P95: 5.0 – 5.4 (n= 13 798)

Reduction in number of live offspring:

not specified

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The male:female ratio was considered unaffected by treatment with the test material up to 25 mg/kg/day. At 25 mg/kg/day, the male ratio was slightly lower than control (44 % vs 54 % for the control group; not statistically significant). As the ratio remained within the range of the historical control data, this was considered not toxicologically relevant.

Historical control data for pregnant Wistar Han rats (period 2016 - 2020):
% Males/Litter mean: 48.9; P5 - P95: 42.7 – 54.7 (n = 6 774)
% Females/Litter mean: 51.1; P5 - P95: 45.3 – 57.3 (n = 7 024)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test material-related effects on litter size of any group.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no test material-related effects on foetal anogenital distance (both sexes) noted after treatment with the test material up to 25 mg/kg/day.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The only external malformation (short tail) was observed in a single foetus of the mid-dose group, this was considered a chance finding. There were no external variations observed in any groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Single foetuses from each of the control, 5 and 25 mg/kg/day groups were found to have malformations. The single occurrences and nature of these findings does not suggest a test material-relationship. Among skeletal variations it was remarkable that there were statistically significantly less foetuses with wavy ribs at 25 mg/kg/day. The reason for this is unknown as wavy ribs are commonly associated with low foetal weights, which were considered unaffected by treatment with test material. Also, less foetuses with wavy ribs is not an adverse effect and considered not toxicologically relevant. Secondly, statistically significantly more foetuses with thoracolumbar short ribs occurred at 25 mg/kg/day. As these short ribs will become part of the lateral processes of the adjacent vertebra, whilst a test material-relationship could not be excluded, these changes were considered not adverse. All other skeletal variations occurred in the absence of a dose-related incidence trend and/or infrequently. Therefore, they were considered not test material-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A single low-dose foetus appeared to have dilatation of both lateral ventricles and had only one eye, while the other was small. This accumulation of malformations in a single foetus without any similar changes at higher doses does not point towards a test material-related effect and therefore was considered a chance finding. Apart from a control foetus with situs inversus, there were no other visceral malformations. Visceral variations that were noted affected the liver (supernumerary lobes), ureter (convoluted and/or dilatation) and kidney (absent papilla). The low incidences and group distribution of these finding did not indicate any test material-relationship.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test material-related effects observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Summary of Clinical Observations

Observation Type: All From Day 2 (Mating (A)) to 21 (Mating)	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Head Tilt
Number of animals affected	0	0	1	0
Number of times recorded	0	0	2	0
% of affected animals	0	0	5	0
First to last seen	-	1	9 - 10	-
Salivation
Number of animals affected	6	11	11	11
Number of times recorded	6	12	23	22
% of affected animals	27	50	50	50
First to last seen	12 -14	9 - 17	9 - 21	9 - 20
Breathing Laboured
Number of animals affected	2	1	1	1
Number of times recorded	2	1	1	1
% of affected animals	9	5	5	5
First to last seen	21 - 21	21 - 21	9 - 9	21 - 21
Breathing, Abnormal Sounds
Number of animals affected	3	4	3	0
Number of times recorded	8	4	7	0
% of affected animals	14	18	14	0
First to last seen	2 - 12	7 - 10	8 - 13	-
Breathing Shallow
Number of animals affected	1	0	0	0
Number of times recorded	2	0	0	0
% of affected animals	5	0	0	0
First to last seen	9 - 12	-	-	-
Hunched Posture
Number of animals affected	0	0	1	8
Number of times recorded	0	0	2	10
% of affected animals	0	0	5	36
First to last seen	-	-	11 - 12	10 - 13
Fur, Erected
Number of animals affected	22	22	21	22
Number of times recorded	73	77	125	152
% of affected animals	100	100	95	100
First to last seen	9 - 21	7 - 20	7 - 21	7 - 20
Fur, Loss
Number of animals affected	2	1	2	1
Number of times recorded	7	4	13	1
% of affected animals	9	5	9	5
First to last seen	15 – 21	15 – 21	9 – 21	20 - 20
Fur, Thin Cover, Ventral Thoracic
Number of animals affected	0	0	1	0
Number of times recorded	0	0	1	0
% of affected animals	0	0	5	0
First to last seen	-	-	12 - 12	-
Skin, Scab Dorsal Cervical
Number of animals affected	1	0	0	0
Number of times recorded	4	0	0	0
% of affected animals	5	0	0	0
First to last seen	9 - 18	-	-	-
Activity Decreased
Number of animals affected	8	7	5	13
Number of times recorded	11	9	5	15
% of affected animals	36	32	23	59
First to last seen	10 – 14	10 – 14	9 – 15	8 - 15

 

Summary of Body Weights

		Days(s) Relative to Mating (Litter: A)
		2	6	9	12	15	18	21
0 mg/kg/day Group 1	Mean	206.2	222.3	225.0	238.6	251.5	2770	307.0
	SD	13.1	14.3	13.6	14.9	15.4	20.1	26.3
	N	22	22	22	22	22	22	21
5 mg/kg/day Group 2	Mean	209.3	225.6	229.8	243.1	254.4	281.8	315.0
	SD	14.7	11.8	127	15.0	15.7	17.5	21.9
	N	21	21	21	21	21	21	21
	% Diff	1.6	1.5	2.1	1.9	1.2	1.7	2.6
10 mg/kg/day Group 3	Mean	207.0	222.5	226.2	241.0	251.1	276.6	306.1
	SD	17.1	17.4	16.4	18.3	22.3	24.5	30.7
	N	22	22	22	22	22	22	22
	% Diff	0.4	0.1	0.5	1.0	-0.1	-0.1	-0.3
25 mg/kg/day Group 4	Mean	210.8	225.6	230.6	243.5	253.2	276.6	299.8
	SD	16.6	18.7	19.5	18.8	19.4	20.2	29.5
	N	22	22	22	22	22	22	22
	% Diff	2.2	1.5	2.5	2.1	0.7	-0.1	-2.4

Anova & Dunnett

 

Summary of Body Weight Gain

		Day(s) Relative to Mating (Litter: A)
		6 – 9 [G]	9 – 12 [G]	12 – 15 [G]	15 – 18 [G]	18 – 21 [G1]	6 – 21 [G]
0 mg/kg/day Group 1	Mean	2.7	13.6	12.9	23.5	30.0	84.5
	SD	5.5	4.8	4.0	7.6	14.3	21.0
	N	22	22	22	22	21	21
5 mg/kg/day Group 2	Mean	4.2	13.3	11.3	27.3	33.3	89.5
	SD	2.9	4.6	4.6	4.8	8.1	14.5
	N	21	21	21	21	21	21
10 mg/kg/day Group 3	Mean	3.7	14.7	10.2	25.5	59.5	83.5
	SD	3.9	4.3	6.9	6.0	13.7	21.3
	N	22	22	22	22	22	22
25 mg/kg/day Group 4	Mean	5.0	12.9	9.7	23.4	23.2	74.1
	SD	5.0	4.2	4.0	6.2	17.1	20.7
	N	22	22	22	22	22	22

[G] - Anova & Dunnett

[G1] - Kruskal-Wallis & Dunn

 

Summary of Gravid Uterine Weights and Corrected Body Weights

		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Bodyweight on Day 6 (g) [G]	Mean	222.5	225.6	222.9	225.6
	SD	14.6	11.8	17.7	18.7
	N	21	21	21	22
	% Diff	-	1.4	0.2	1.4
Terminal body weight (g) [G]	Mean	307.0	315.0	307.9	299.8
	SD	26.3	21.9	30.3	29.5
	N	21	21	21	22
	% Diff	-	2.6	0.3	-2.4
Gravid uterus weight (g) [G]	Mean	68.12	72.75	73.63	68.94
	SD	10.84	11.71	9.12	14.53
	N	21	21	21	22
	% Diff	-	6.80	8.09	1.20
Adjusted BWG (6-abw) (g) [G]	Mean	16.40	16.72	11.32	5.20*
	SD	15.2	8.85	16.34	17.97
	N	21	21	21	22
	% Diff	-	1.94	-30.97	-68.30

[G] = Anova and Dunnett

*= p ≤ 0.05

 

Summary of Food Consumption: Food Mean Daily Consumption (g/animal/day)

		Day(s) Relative to Mating (Litter: A)
		6 – 9	9 – 12	12 – 15	15 – 18	18 – 21	6 – 21
0 mg/kg/day Group 1	Mean	16.11	16.80	19.42	1+.83	17.47	17.93
	SD	2.58	1.79	2.83	2.78	4.06	1.99
	N	22	22	22	22	22	22
5 mg/kg/day Group 2	Mean	15.25	16.41	19.32	19.65	18.44	17.82
	SD	3.53	2.10	2.79	2.01	2.52	1.78
	N	21	21	21	21	21	21
	% Diff	-5.29	-2.32	-0.55	-0.92	5.58	-0.62
10 mg/kg/day Group 3	Mean	15.44	16.33	18.79	18.64	16.88	17.21
	SD	1.84	2.48	2.68	2.56	4.45	2.20
	N	22	22	22	22	22	22
	% Diff	-4.14	-2.80	-3.43	-6.04	-3.38	-4.01
25 mg/kg/day Group 4	Mean	14.98	15.61	17.71	18.02	15.30	16.32*
	SD	2.15	1.69	2.23	1.99	4.26	1.62
	N	22	22	22	22	22	22
	% Diff	-6.96	-7.17	-8.81	-9.17	-12.40	-8.98

Anova and Dunnett

*= p ≤ 0.05

 

Summary of Thyroid Hormone Values

		T3 (ng/mL) [G]	TSH mU/L [G]	T4 Ng/mL [G]
0 mg/kg/day Group 1	Mean	0.368	0.4264	21.39
	SD	0.101	0.3230	6.11
	N	20	21	20
5 mg/kg/day Group 2	Mean	0.330	0.3275	21.12
	SD	0.065	0.1422	4.64
	N	21	21	21
	tCtrl	0.90	0.77	0.99
10 mg/kg/day Group 3	Mean	0.334	0.4800	18.13
	SD	0.132	0.2682	6.40
	N	22	22	22
	tCtrl	0.91	1.13	0.85
25 mg/kg/day Group 4	Mean	0.338	0.5285	19.68
	SD	0.092	0.2725	4.94
	N	22	22	22
	tCtrl	0.92	1.24	0.92

[G] Anova & Dunnett

 

Summary of Absolute Organ Weights

		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Terminal body weight (g) [G]	Mean	307.0	315.0	306.1	299.8
	SD	26.3	21.9	30.7	29.5
	N	21	21	22	22
	% Diff	-	2.6	-0.3	-2.4
Gland, Thyroid weight (g) [G]	Mean	0.01340	0.01436	0.01303	0.01343
	SD	0.00262	0.00263	0.00361	0.00234
	N	21	21	21	22
	% Diff	-	7.21649	-2.70174	0.27308
Liver weight (g) [G]	Mean	13.0171	13.6009	12.7425	13.2545
	SD	19352	1.4204	2.1100	2.1357
	N	21	21	21	22
	% Diff	-	4.4849	-2.1093	1.8234
Thymus weight (g) [G]	Mean	0.1927	0.2158	0.1828	0.1608*
	SD	0.0476	0.0486	0.0428	0.0332
	N	21	21	21	22
	% Diff	-	12.0119	-5.1162	-16.5539

[G] = Anova & Dunnett

* = p ≤ 0.05

 

Summary of Organ Weights Relative to Body Weight

		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Gland, thyroid (%) [G]	Mean	0.00438	0.00456	0.00422	0.00448
	SD	0.00089	0.00076	0.00107	0.00066
	N	21	21	21	22
	% Diff	-	3.96415	-3.65116	2.12505
Liver (%) [G]	Mean	4.22858	4.32236	4.12195	4.40808
	SD	0.39564	0.39046	0.35029	0.45987
	N	21	21	21	22
	% Diff	-	2.21772	-2.52159	4.24496
Thymus (%) [G]	Mean	0.06288	0.06849	0.05933	0.05353
	SD	0.01546	0.01500	0.01206	0.00987
	N	21	21	21	22
	% Diff	-	8.93131	-564677	-14.87019

[G] = Anova & Dunnett

 

Summary of Macroscopic Pathology: Terminal Euthanasia

Incidence	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of animals	21	22	21	22
Gland, Thyroid
Submitted	21	22	21	22
No visible lesion	19	22	20	21
Small	1	0	1	1
Enlargement	2	0	0	0
Large Intestine, Cecum
Submitted	0	0	0	1
Abnormal appearance	.	.	.	1
Large Intestine, Colon
Submitted	0	0	0	1
Abnormal appearance	.	.	.	1
Large Intestine, Rectum
Submitted	0	0	0	1
No visible lesion	.	.	.	1
Liver
Submitted	21	22	21	22
No visible lesion	19	22	20	22
Discoloration, dark; red	1	0	0	0
Focus, pale; red, lateral lobe	1	0	1	0
Focus, pale; red, medial lobe	1	0	1	0
Small Intestine, Duodenum
Submitted	0	0	0	1
Abnormal appearance	.	.	.	1
Small Intestine, Ileum
Submitted	0	0	0	1
Abnormal appearance	.	.	.	1
Small Intestine, Jejunum
Submitted	0	0	0	1
Abnormal appearance	.	.	.	1
Stomach
Submitted	0	0	0	1
No visible lesion	.	.	.	1
Thymus
Submitted	21	22	21	22
No visible lesion	21	22	21	21
Small	0	0	0	1

 

Summary of Macroscopic Pathology: Delivered

Incidence	0 mg/kg/day Group 1	10 mg/kg/day Group 3
Number of animals	1	1
Gland, Thyroid
Submitted	1	1
No visible lesion	1	1
Submitted	1	1
No visible lesion	1	1
Thymus
Submitted	1	1
No visible lesion	1	1

 

Summary of Macroscopic Pathology: Terminal Euthanasia

Incidence	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of animals	21	22	21	22
Gland, Thyroid
Examined	21	22	21	22
No visible lesions	20	22	18	19
Hypertrophy, follicular cell	1	0	1	1
…Minimal	1	0	1	1
Ectopia, thymic	0	0	2	2
…Minimal	0	0	2	2

 

Summary of Macroscopic Pathology: Delivered

Incidence	0 mg/kg/day Group 1	10 mg/kg/day Group 3
Number of animals	1	1
Gland, Thyroid
Examined	1	1
No visible lesions	0	1
Hypertrophy, follicular cell	1	0
…Minimal	1	0

 

Summary of Maternal Performance and Mortality

Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Group size - females		22	22	22	22
Number of females pregnant [f]	N+ve	22	21	22	22
	%	100.0	95.5	100.0	100.0
Females with live foetuses [f]	N+ve	22	21	22	22
	%	100.0	100.0	100.0	100.0
Total resorptions [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Females with all nonviable [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Terminal euthanasia [f]	N+ve	21	22	21	22
	%	95.5	100.0	95.5	100.0
Unscheduled death / euthanasia [f]	N+ve	1	0	1	0
	%	4.5	0.0	4.5	0.0
Delivered [f]	N+ve	1	0	1	0
	%	4.5	0.0	4.5	0.0

[f] Fisher’s exact

 

Summary of Ovarian and Uterine Examinations and Litter Observations

Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Female with live foetuses	N+ve	21	21	21	22
	%	100.0	100.0	100.0	100.0
Number of corpora lutea [k]	Mean	11.5	11.7	12.2	11.3
	SD	2.0	1.7	2.0	1.2
	N	21	21	21	22
	% Diff	-	2.1	6.2	-1.8
Number of implantations [k]	Mean	10.6	10.5	11.2	10.2
	SD	1.9	1.9	1.5	2.2
	N	21	21	21	22
	% Diff	-	-0.9	5.8	-3.7
Pre-implantation loss (%) [k]	Mean	7.00	9.31	6.86	9.06
	SD	8.64	14.66	9.63	16.99
	N	21	21	21	22
	% Diff	-	32.98	-2.02	29.38
Total number of foetuses [k]	Mean	10.4	10.3	10.8	10.0
	SD	2.0	1.9	1.2	2.3
	N	21	21	21	22
	% Diff	-	-1.4	3.2	-4.5
Number of live foetuses [k]	Mean	10.4	10.3	10.8	10.0
	SD	2.0	1.9	1.2	2.3
	N	21	21	21	22
	% Diff	-	-1.4	3.2	-4.5
Number of dead foetuses [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	21	21	21	22
	% Diff	-	-	-	-
Number of early resorptions [k]	Mean	0.2	0.2	0.5	0.3
	SD	0.5	0.7	0.7	0.5
	N	21	21	21	22
	% Diff	-	25.0	150.0	43.2
Number of late resorptions [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	21	21	21	22
	% Diff	-	-	-	-
Total number of resorptions [k]	Mean	0.2	0.2	0.5	0.3
	SD	0.5	0.7	0.7	0.5
	N	21	21	21	22
	% Diff	-	25.0	150.0	43.2
Post-implantation loss (%) [k]	Mean	1.90	2.17	382	2.92
	SD	5.12	6.79	5.98	5.11
	N	21	21	21	22
	% Diff	-	13.69	100.61	53.43
Number of live male foetuses [k]	Mean	5.7	5.5	5.4	4.4
	SD	2.1	1.7	1.7	2.0
	N	21	21	21	22
	% Diff	-	-3.4	-5.0	-23.0
Number of live female foetuses [k]	Mean	4.8	4.8	5.4	5.6
	SD	1.9	1.7	2.0	2.2
	N	21	21	21	22
	% Diff	-	1.0	13.0	17.4
Live male foetus/litter (%) [k]	Mean	53.98	53.11	50.32	44.02
	SD	18.59	14.01	15.73	19.60
	N	21	21	21	22
	% Diff	-	-1.61	-6.78	-18.45
Live female foetus/litter (%) [k]	Mean	46.02	46.89	49.68	55.98
	SD	18.59	14.01	15.73	19.60
	N	21	21	21	22
	% Diff	-	1.89	7.96	21.64
Mean foetal weight males (g) [G]	Mean	5.041	5.463**	5.261	5.304
	SD	0.539	0.265	0.465
	N	21	21	21	21
	% Diff	-	8.384	4.375	5.224
Mean foetal weight females (g) [G]	Mean	4.845	5.242**	5.011	4.996
	SD	0.534	0.301	0.363	0.427
	N	21	21	21	22
	% Diff	-	8.205	3.435	3.136
Mean foetal weight all (g) [G]	Mean	4.949	5.358**	5.132	5.148
	SD	0.513	0.282	0.348	0.430
	N	21	21	21	22
	% Diff	-	8.279	3.703	4.025
Live male foetuses pl. wt. (mean) [G1]	Mean	0.428	0.452	0.420	0.423
	SD	0.064	0.055	0.055	0.054
	N	21	21	21	21
	% Diff	-	5.596	-1.770	-1.227
Live female foetuses pl. wt. (mean) [G1]	Mean	0.401	0.423	0.404	0.397
	SD	0.053	0.053	0.059	0.039
	N	21	21	21	22
	% Diff	-	5.600	0.702	-0.873
Mean placental weight (g) [G]	Mean	0.414	0.438	0.413	0.405
	SD	0.053	0.049	0.054	0.039
	N	21	21	21	22
	% Diff	-	5.869	-0.121	-1.993
Mean foetal AGD males (mm) [G]	Mean	2.60	2.74	2.69	2.72
	SD	0.22	0.28	0.31	0.27
	N	21	21	21	21
	% Diff	-	5.52	3.44	4.63
Mean foetal AGD females (mm) [G]	Mean	1.10	1.22	1.17	1.16
	SD	0.17	0.24	0.21	0.14
	N	21	21	21	22
	% Diff	-	11.38	6.355	5.50
Mean normalised foetal AGD m [G]	Mean	1.518	1.560	1.547	1.562
	SD	0.103	0.160	0.165	0.161
	N	21	21	21	21
	% Diff	-	2.753	1.905	2.933
Mean normalised foetal AGD f [G]	Mean	0.651	0.704	0.685	0.680
	SD	0.095	0.138	0.122	0.085
	N	21	21	21	22
	% Diff	-	8.200	5.280	4.407

[G] - Anova & Dunnett

** = p ≤ 0.01

[G1] - Anova & Dunnett

[k] - Kruskal-Wallis & Dunn

 

Summary of Foetal Abnormalities by Finding

External Examination		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined		219	216	226	219
Number of foetuses evaluated		219	216	226	219
Number of litters examined		21	21	21	22
Number of litters evaluated		21	21	21	22
Tail
Tail, short - malformation	Foetuses N (%)	0 (0.00)	0 (0.00)	1 (0.40)	0 (0.00)
	Litters N (%)	0 (0.00)	0 (0.00)	1 (4.8)	0 (0.00)

[Foetuses %] - Kruskal-Wallis & Dunn Foetuses N(%) N=Group Foetal Incidence;(%)=Mean Litter % of Foetuses with the Abnormality

 

Summary of Foetal Abnormalities by Finding

Fixed Head		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined		110	107	112	110
Number of foetuses evaluated		19	216	226	219
Number of litters examined		21	21	21	22
Number of litters evaluated		21	21	21	22
Brain
Lateral ventricle, Both, Dilatation, Severe - Malformation	Foetuses N (%)	0 (0.00)	1 (0.79)	0 (0.00)	0 (0.00)
	Litters N (%)	0 (0.00)	1 (4.8)	0 (0.00)	0 (0.00)
Eye
Eye, Left, Small - Malformation	Foetuses N (%)	0 (0.00)	1 (0.79)	0 (0.00)	0 (0.00)
	Litters N (%)	0 (0.00)	1 (4.8)	0 (0.00)	0 (0.00)
Eye, Right, Absent - Malformation	Foetuses N (%)	0 (0.00)	1 (0.79)	0 (0.00)	0 (0.00)
	Litters N (%)	0 (0.00)	1 (4.8)	0 (0.00)	0 (0.00)

[Foetuses %] - Kruskal-Wallis & Dunn FoetusesN(%) N=Group Foetal Incidence;(%)=Mean Litter % of Foetuses with the Abnormality

 

Summary of Foetal Abnormalities by Finding

Fresh Visbody		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined		110	107	112	110
Number of foetuses evaluated		219	216	226	219
Number of litters examined		21	21	21	22
Number of litters evaluated		21	21	21	22
General
General, Situs inversus - Malformation	Foetuses N (%)	1 (0.95)	0 (0.00)	0 (0.00)	0 (0.00)
	Litters N (%)	1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)
Kidney
Renal papilla, Left, Absent - Variation	Foetuses N (%)	0 (0.00)	1 (0.95)	0 (0.00)	0 (0.00)
	Litters N (%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Renal papilla, right, Absent - Variation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	2 (1.82)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Liver
Lobe, Left medial, Supernumerary - Variation	Foetuses N (%)	5 (4.30)	6 (5.08)	8 (6.92)	6 (5.38)
	Litters N (%)	5 (23.8)	4 (19.0)	7 (33.3)	4 (18.2)
Lobe, Right medial, Supernumerary - Variation	Foetuses N (%)	2 (1.75)	2 (2.14)	4 (3.81)	4 (3.48)
	Litters N (%)	2 (9.5)	2 (9.5)	3 (14.3)	4 (18.2)
Spleen
Spleen, Discoloured - Incidental	Foetuses N (%)	1 (0.79)	0 (0.00)	0 (0.00)	0 (0.00)
	Litters N (%)	1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)
Ureter
Ureter, Both, Dilatation, Minimal - Variation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.91)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Ureter, Left, Convoluted - Variation	Foetuses N (%)	0 (0.00)	2 (1.90)	0 (0.00)	0 (0.00)
	Litters N (%)	0 (0.0)	2 (9.5)	0 (0.0)	0 (0.0)
Ureter, Right, Convoluted - Variation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.91)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Ureter, Right, Dilatation, Minimal - Variation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.91)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)

 

Summary of Foetal Abnormalities by Finding

Skeletal		0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined		109	109	114	109
Number of foetuses evaluated		219	216	226	219
Number of litters examined		21	21	21	22
Number of litters evaluated		21	21	21	22
Forelimb
Humerus, Right, Bent - Malformation	Foetuses N (%)	0 (0.00)	1 (0.79)	0 (0.00)	0 (0.00)
	Litters N (%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Metacarpal, 1 or more, Unossified - Variation	Foetuses N (%)	7 (5.71)	0 (0.00)	0 (0.00)	0 (0.00)
	Litters N (%)	2 (9.5)	0 (0.0)	0 (0.0)	0 (0.0)
Pelvic girdle
Ilium, Both, Misaligned - Variation	Foetuses N (%)	3 (2.38)	6 (5.40)	4 (3.33)	3 (2.73)
	Litters N (%)	2 (9.5)	4 (19.0)	3 (14.3)	3 (13.6)
Rib
Costal cartilage, 1 or more, Fused - Variation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (2.27)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Rib, 1 or more, Wavy rib - Variation	Foetuses N (%)	30 (27.59)	24 (22.47)	18 (16.03)	4 (3.48)**
	Litters N (%)	14 (66.7)	11 (52.4)	8 (38.1)	4 (18.2)
Scapula
Scapula, Right, Bent - Variation	Foetuses N (%)	0 (0.00)	1 (0.79)	1 (0.95)	0 (0.00)
	Litters N (%)	0 (0.0)	1 (4.8)	1 (4.8)	0 (0.0)
Skull
Frontal, Both, Incomplete ossification - Variation	Foetuses N (%)	1 (0.95)	0 (0.00)	1 (0.79)	2 (1.52)
	Litters N (%)	1 (4.8)	0 (0.0)	1 (4.8)	1 (4.5)
Interparietal, Incomplete ossification - Variation	Foetuses N (%)	2 (1.90)	2 (1.59)	4 (3.97)	2 (1.52)
	Litters N (%)	2 (9.5)	2 (9.5)	3 (14.3)	1 (4.5)
Parietal, Both, Incomplete ossification - Variation	Foetuses N (%)	2 (1.90)	0 (0.00)	0 (0.00)	1 (0.76)
	Litters N (%)	2 (9.5)	0 (0.0)	0 (0.0)	1 (4.5)
Parietal, Left, Incomplete ossification - Variation	Foetuses N (%)	0 (0.00)	0 (0.00)	1 (1.19)	0 (0.00)
	Litters N (%)	0 (0.0)	0 (0.0)	1 (4.8)	0 (0.0)
Parietal, Right, Incomplete ossification - Variation	Foetuses N (%)	1 (0.95)	0 (0.00)	0 (0.00)	2 (1.52)
	Litters N (%)	1 (4.8)	0 (0.0)	0 (0.0)	1 (4.5)
Sternebra
Sternebra, 1 or more, Misaligned - Variation	Foetuses N (%)	3 (2.78)	1 (0.79)	1 (0.79)	1 (0.91)
	Litters N (%)	3 (14.3)	1 (4.8)	1 (4.8)	1 (4.5)
Sternebra, 1 or more, Supernumerary - Malformation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.91)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Sternebra, 1 or more, Unossified - Variation	Foetuses N (%)	6 (4.92)	0 (0.00)	1 (0.95)	0 (0.00)
	Litters N (%)	2 (9.5)	0 (0.0)	1 (4.8)	0 (0.0)
Sternebra, 1 or more, Incomplete ossification - Variation	Foetuses N (%)	1 (0.95)	1 (0.79)	1 (0.79)	1 (0.76)
	Litters N (%)	1 (4.8)	1 (4.8)	1 (4.8)	1 (4.5)
Supernumerary rib
Cervical, 1 or more, Short - Variation	Foetuses N (%)	9 (9.21)	5 (5.24)	6 (4.92)	8 (10.08)
	Litters N (%)	7 (33.3)	3 (14.3)	5 (23.8)	6 (27.3)
Thoracolumbar, 1 or more, Full - Variation	Foetuses N (%)	6 (5.32)	10 (9.52)	4 (3.33)	8 (6.63)
	Litters N (%)	4 (19.0)	6 (28.6)	4 (19.0)	6 (27.3)
Thoracolumbar, 1 or more, Short - Variation	Foetuses N (%)	49 (44.09)	64 (57.85)	61 (52.30)	78 (71.40)**
	Litters N (%)	17 (81.0)	21 (100.0)	21 (100.0)	22 (100.0)
Vertebra
Cervical vertebra, 1 or more, Supernumerary - Malformation	Foetuses N (%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.91)
	Litters N (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Lumbar vertebra, 1 or more, Supernumerary - Malformation	Foetuses N (%)	1 (1.59)	0 (0.00)	0 (0.00)	0 (0.00)
	Litters N (%)	1.48	0 (0.0)	0 (0.0)	0 (0.0)
Thoracic centrum, 1 or more, Incomplete ossification - Variation	Foetuses N (%)	0 (0.00)	3 (2.70)	0 (0.00)	1 (0.91)
	Litters N (%)	0 (0.0)	3 (14.3)	0 (0.0)	1 (4.5)

[Foetuses %] - Kruskal-Wallis & Dunn

Foetuses N (%) N = Group Foetal Incidence; (%) = Mean Litter % of Foetuses with the Abnormality

** = p ≤ 0.01

 

Summary of Foetal Abnormalities by Classification

External	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined	219	216	226	219
Number of foetuses evaluated	219	216	226	219
Number of litters examined	21	21	21	22
Number of litters evaluated	21	21	21	22
Malformation
Number of foetuses	0	0	1	0
Litter % of foetuses [k]	0.00	0.00	0.40	0.00
Number of litters	0	0	1	0
All classifications
Number of foetuses	0	0	1	0
Litter % of foetuses [k]	0.00	0.00	0.40	0.00
Number of litters	0	0	1	0

[k] - Kruskal-Wallis & Dunn

 

Summary of Foetal Abnormalities by Classification

Fixed Head	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined	110	107	112	110
Number of foetuses evaluated	219	216	226	219
Number of litters examined	21	21	21	22
Number of litters evaluated	21	21	21	22
Malformation
Number of foetuses	0	1	0	0
Litter % of foetuses [k]	0.00	0.79	0.00	0.00
Number of litters	0	1	0	0
All classifications
Number of foetuses	0	1	0	0
Litter % of foetuses [k]	0.00	0.79	0.00	0.00
Number of litters	0	1	0	0

[k] - Kruskal-Wallis & Dunn

 

Summary of Foetal Abnormalities by Classification

Fresh VisBody	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined	110	107	112	110
Number of foetuses evaluated	219	216	226	219
Number of litters examined	21	21	21	22
Number of litters evaluated	21	21	21	22
Incidental
Number of foetuses	1	0	0	0
Litter % of foetuses [k]	0.97	0.00	0.00	0.00
Number of litters	1	0	0	0
Variation
Number of foetuses	7	10	12	11
Litter % of foetuses [k]	6.04	9.13	10.73	9.77
Number of litters	7	8	8	6
Malformation
Number of foetuses	1	0	0	0
Litter % of foetuses [k]	0.95	0.00	0.00	0.00
Number of litters	1	0	0	0
All classifications
Number of foetuses	9	10	12	11
Litter % of foetuses [k]	7.79	9.13	10.73	9.77
Number of litters	9	8	8	6

[k] - Kruskal-Wallis & Dunn

 

Skeletal	0 mg/kg/day Group 1	5 mg/kg/day Group 2	10 mg/kg/day Group 3	25 mg/kg/day Group 4
Number of foetuses examined	109	109	114	109
Number of foetuses evaluated	219	216	226	219
Number of litters examined	21	21	21	22
Number of litters evaluated	21	21	21	22
Variation
Number of foetuses	73	83	75	88
Litter % of foetuses [k]	66.52	75.59	65.16	81.59
Number of litters	20	21	21	22
Malformation
Number of foetuses	1	1	0	1
Litter % of foetuses [k]	1.59	0.79	0.00	0.91
Number of litters	1	1	0	1
All classifications
Number of foetuses	74	83	75	89
Litter % of foetuses [k]	68.11	75.59	65.16	82.50
Number of litters	20	21	21	22

[k] - Kruskal-Wallis & Dunn

 

 

 

Historical Control Data

Rat: Crl:WI(Han) (outbred. SPF-Quality)

Gestation Day 21

Study Date Range: 2016 - 2020

No. of Studies: 60

	Mean	SD	Median	Min.	Max.	P5	P95	Foetuses	Litters
Malformations
Total external malformations								20	20
Total visceral malformations								28	28
Total skeletal malformations								74	67
Total malformations								107	97
External
Tail absent	0.0	0.05	0.0	0.0	0.4	0.0	0.0	1	1
Visceral
Eye(s)- absent and/or Small	0.1	0.71	0.0	0.0	5.3	0.0	0.9	4	4
Hydrocephaly- internal	0.0	0.15	0.0	0.0	0.9	0.0	0.	2	2
Situs inversus	0.2	0.39	0.0	0.0	1.5	0.0	1.1	15	15
Skeletal
Limb bone(s)- bent	0.4	0.69	0.0	0.0	2.3	0.0	2.3	15	13
Sternal anomaly	0.0	0.13	0.0	0.0	0.7	0.0	0.0	2	2
Vertebral anomaly with or without associated rib anomaly	0.3	0.47	0.0	0.0	1.7	0.0	1.4	20	20
Variations
Skeletal
14th rudimentary rib(s)	53.7	6.25	52.7	41.8	71.8	44.6	67.0	3 691	1 190

 

Conclusions:

Under the conditions of the study, the NOAELs for the test material were:
Maternal toxicity: 10 mg/kg/day, based on decreased food consumption and lower mean body weight gain adjusted for gravid uterus.
Developmental toxicity: At least 25 mg/kg/day (no test material-related adverse findings).

Executive summary:

The potential of the test material to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterise maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive, was assessed according to OECD Test Guideline 414 and in compliance with GLP.

The No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 5, 10 and 25 mg/kg/day, based on the results of the Dose Range Finding Study (2 groups, 25 and 50 mg/kg/day). The study design was as follows:

Group 1: 0 mg/kg/day (vehicle only)

Group 2: 5 mg/kg/day

Group 3: 10 mg/kg/day

Group 4: 25 mg/kg/day

Dose volume was 5 mL/kg and there were 22 females per dose group.

Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the maternal animals (F0-generation): Mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic examination, organ weights, uterine contents, microscopic examination (thyroid gland), corpora lutea, implantation sites, and pre- and post-implantation loss.

In addition, the following parameters were determined for the foetal offspring (F1-generation): The number of live and dead foetuses, foetal body weights, sex ratio, anogenital distance, placenta weights, external, visceral and skeletal malformations and developmental variations.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No mortality occurred during the study period.

At 25 mg/kg/day, mean food consumption taken over the total treatment period was decreased (9 % lower than control), as well as a decrease in body weight between Days 18 - 21 post-coitum (2.4 % lower than control) and a markedly lower mean body weight gain when adjusted for gravid uterus weight (68 % lower than control). These findings were considered adverse.

Thymus weights at this dose were lower than control. However, in the absence of microscopic evaluation, no assessment of adversity could be made.

No test material-related or toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. clinical appearance, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (liver, thyroid gland), macroscopic evaluation, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).

No test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, foetal body weights, anogenital distance, placenta weights, external, visceral and skeletal malformations and developmental variations).

Under the conditions of the study, the NOAELs for the test material were:

Maternal toxicity:10 mg/kg/day, based on decreased food consumption and lower mean body weight gain adjusted for gravid uterus.

Developmental toxicity: At least 25 mg/kg/day (no test material-related adverse findings).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was conducted according to standardised guidelines and in compliance with GLP in an appropriate test species.

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

Key Study: Langedijk (2021)

The potential of the test material to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterise maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive, was assessed according to OECD Test Guideline 414 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 5, 10 and 25 mg/kg/day, based on the results of the Dose Range Finding Study (2 groups, 25 and 50 mg/kg/day). The study design was as follows:

Group 1: 0 mg/kg/day (vehicle only)

Group 2: 5 mg/kg/day

Group 3: 10 mg/kg/day

Group 4: 25 mg/kg/day

Dose volume was 5 mL/kg and there were 22 females per dose group.

Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the maternal animals (F0-generation): Mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic examination, organ weights, uterine contents, microscopic examination (thyroid gland), corpora lutea, implantation sites, and pre- and post-implantation loss.

In addition, the following parameters were determined for the foetal offspring (F1-generation): The number of live and dead foetuses, foetal body weights, sex ratio, anogenital distance, placenta weights, external, visceral and skeletal malformations and developmental variations.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No mortality occurred during the study period.

At 25 mg/kg/day, mean food consumption taken over the total treatment period was decreased (9 % lower than control), as well as a decrease in body weight between Days 18 - 21 post-coitum (2.4 % lower than control) and a markedly lower mean body weight gain when adjusted for gravid uterus weight (68 % lower than control). These findings were considered adverse.

Thymus weights at this dose were lower than control. However, in the absence of microscopic evaluation, no assessment of adversity could be made.

No test material-related or toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. clinical appearance, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (liver, thyroid gland), macroscopic evaluation, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).

No test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, foetal body weights, anogenital distance, placenta weights, external, visceral and skeletal malformations and developmental variations).

Under the conditions of the study, the NOAELs for the test material were:

Maternal toxicity: 10 mg/kg/day, based on decreased food consumption and lower mean body weight gain adjusted for gravid uterus.

Developmental toxicity: At least 25 mg/kg/day (no test material-related adverse findings).

 

Supporting Study: Langedijk (2021) Range-finder

The dose range-finding study was conducted to determine the highest tolerated dose level of the test material orally by gavage in pregnant rats. This study was performed as a Dose Range Finding study for the subsequent Prenatal Developmental Toxicity study by oral gavage and as such was not conducted to a guideline or GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Timed-pregnant Wistar-Han rats were administered the test material at a dose of either 25 or 50 mg/kg/day by oral gavage from Day 6 up to and including Day 20 post-coitum.  At intervals throughout the day, mortality and cage side clinical observations were recorded. Every third day during treatment and on the day of necropsy, detailed clinical observations, body weights, food consumption were recorded.  Following termination on Day 21 (group 1) or Day 15 post-coitum (early termination) external, thoracic and abdominal examinations were made, with special attention being paid to the reproductive organs. Number of corpora lutea, weight of the liver, thymus and uterus, placental weights of live foetuses, number of implantation sites, number and distribution of live and dead foetuses, number and distribution of early and late resorptions were determined. External examination, external sex determination and body weight were made of live foetuses of dams surviving until scheduled necropsy on Day 21 post-coitum.

A dose of 50 mg/kg/day was clearly in excess of the maximum tolerated dose, being associated with severe body weight loss and low food consumption, necessitating the early termination of the group. The dose level of 25 mg/kg bw/day was associated with clear but tolerable maternal toxicity in terms of clinical signs, reduced body weight and body weight gain, lower food intake, lower absolute and relative thymus weight with macroscopic correlate. Therefore, it was considered that a dose of 25 mg/kg/day would adequately demonstrate systemic toxicity in accordance with the OECD 414 test guideline requirements. Dose levels of 3 and 10 mg/kg/day were selected on the expectation they would characterise a dose-response, with the lowest dose expected to be a No Adverse Effect Level (NOAEL).

 

Disregarded Study: Manjunath (2021)

The objective of this study was to assess the effects of exposure to the test material on the presumed pregnant rats and in the developing organisms, including an assessment of maternal effects as well as death, structural abnormalities, or altered growth in the foetus. The study was awarded a reliability score of 3 in accordance with the criteria set forth by Klimisch et al. (1997).

A total of 100 presumed pregnant female Sprague Dawley rats were allocated to four groups. Each group consisted of 25 presumed-pregnant female rats (Dams). The test material was administered by oral gavage, at a dose volume of 10 mL/kg body weight to each animal, with the doses of 0, 5, 15 and 50 mg/kg body weight for groups G1 (Vehicle Control), G2 (Low Dose), G3 (Mid Dose) and G4 (High Dose), respectively from Gestation Day (GD) 5 to 19.

A total of 22, 21, 21 and 22 presumed pregnant female were confirmed as pregnant (with evidence of implantations) across groups, yielding to pregnancy rates of 88 %, 84 %, 84 % and 88 % at the time of caesarean section from the groups G1, G2, G3 and G4, respectively. Among these, a total of 22, 21, 20 and 20 presumed pregnant females from group G1, G2, G3 and G4, respectively were found with live fetuses.

Upon review of the data by the Sponsor, the study was terminated without finalization of study report or interpretation of the data as the data was considered as not valid by sponsor.

Justification for classification or non-classification

The key data on DMTE showed no test material-related or toxicologically significant changes were noted in any of the developmental parameters investigated. However, in accordance with Annex VI, Regulation (EC) No 1272/2008, the substance has a harmonised classification with respect to reproduction as Category 2 (H361d; Suspected of damaging fertility or the unborn child).