[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Read-across on racemic form of Cycloalkane ethers:

• Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test: NOAEL Fertility = 800 mg/kg bw/d (OECD 422, GLP, K, Rel. 1)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2008-09-29 to 2009-08-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 422 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD GLP (inspected on 05th to 09th and 26th to 30th November 2007, Signed on 12th November 2008)

Limit test:

no

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 44414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 284 to 318 g; Females: 178 to 214 g
- Fasting period before study: none
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular oestrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 31/08).
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with music during the light period

IN-LIFE DATES: From: 2008-09-28 To: 2009-08-27

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added.
Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 24897436

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day, one sample (middle) from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analysed by GC coupled to an FI detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

The identity of the test material was confirmed by its retention time, which was similar to that measured in the working standards. The application formulations investigated during the study were found to comprise the test material in the range of 91.7% to 108.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the test material in the preparations was approved because single results did not deviate more than 5.9% (<15%) from the corresponding mean. The application formulations were considered to be stable for at least 7 days when kept at room temperature.

Duration of treatment / exposure:

Males: minimum 4 weeks. Females: approximately 7 weeks.

Frequency of treatment:

Daily

Details on study schedule:

See Table 7.8.1/1

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C05595, using dose levels of 100, 300, and 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Positive control:

None

Parental animals: Observations and examinations:

VIABILITY / MORTALITYS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Daily cage-side clinical observations (once daily during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, performed outside the home cage. Animals were observed
- Parameters examined: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1-8, 8-14 and 14-16; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 animals/sex/dose
- Parameters checked in table 5.8.1/2.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals:5 animals/sex/dose
- Parameters checked in table 5.8.1/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males shortly before the scheduled sacrifice and females on day 3 or 4 post partum
- Dose groups that were examined: five P generation males and five P generation females from each group (erroneously six females were investigated in group 4)
- Battery of functions tested:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and faecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or faeces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in male parental animals: testis weight, epididymis weight. The testes were stained by PAS hematoxylin for qualitative sperm staging.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS:
No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects.
- Maternal animals: All surviving animals were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

GROSS PATHOLOGY: Yes (see table 7.8.1/3)

HISTOPATHOLOGY: Yes (see table 7.8.1/3)

The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

Postmortem examinations (offspring):

SACRIFICE
Pups were sacrificed on day 4 post partum

GROSS NECROPSY
- All pups were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:

The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied to the macroscopical findings.

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All males and females treated with 400 or 800 mg/kg bw/d pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. This was considered to be a sign of discomfort following treatment rather than a toxic effect of the test item. One male and one female treated with 800 mg/kg bw/d had salivation for isolated days. In addition, one female had salivation for most of the prepairing, pairing and gestation periods. This was considered to be a sign of discomfort following the treatment.
Other clinical signs noted were a wound on the shoulder of one male in low-dose group and hair loss in one dam in the control group. These findings were considered to be incidental.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Normal responses to the taste of fragrances.

- Males: In high-dose group, body weight gain was statistically significantly reduced on days 3, 6 and 10 of the pre-pairing period and on the last day of the after pairing period. However, mean body weight was similar in all dose groups throughout the study. No test item-related effects were noted in the other groups.
- Females: In high-dose group 4, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight on days 3, 4 and 6 during the pre-pairing period.This was considered to be a result of treatment with the test item. No other test item-related effects were noted during the study at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Normal responses to the taste of fragrances.

- Males: Mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period (-5.6% and -6.1%, respectively). Thereafter, food consumption was not affected by treatment with the test item in any dose group.
- Females: In high-dose group, mean food consumption was statistically significantly reduced over the first week of the pre-pairing period (-18.4% compared to the control group). This reduction was considered to be a test item-related effect. The statistically significant increase in low-dose group in the first week of the pre-pairing period was considered to be incidental due to the lack of a dose-dependent pattern.
No other test item-related effects were noted in the food consumption during the study at any dose level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Males The level of platelets was statistically significantly increased in medium-dose groups (+23.2%, compared to the control group) and 4 (+33.8%) and was outside the historical control data in high-dose group.
- Females: In the females in high-dose group, although the prothrombin time was not statistically significantly increased (+15.3%), the level was outside the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Males The level of cholesterol was statistically significantly increased in medium- and high-dose dose groups (+28.5% and +44.0%, respectively). Both these values were outside the range of the historical control data. The level of protein (+5.3% in medium-dose group and +8.6% in high-dose group) as well as globulin (+9.6% in medium-dose group and +16.5% in high-dose group) were increased and although the values were not statistically significant, they were outside the range of the historical control data.
- Females: The level of cholesterol in medium- and high-dose groups (+59.0% and +125.5%, respectively) was outside the range of the historical control data and was statistically significantly increased in high-dose group. The level of globulin was statistically significantly increased in medium- and high-dose groups (+17.3% and +16.1%, respectively) and both levels were outside the range of the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In high-dose group, there was an increased incidence in males of a reduced number of rearings (80%) compared to the control group (20%). In addition, body temperature was statistically significantly decreased in males (37.9 °C compared to the control group 38.5 °C) and females (38.3 °C compared to 39.0 °C in the control group).
In medium-dose group, the body temperature of the females was statistically significantly reduced (38.5 °C compared to 39.0 °C in the control group).
Although the body temperatures were within the range of the historical control data, since they decreased in a dose-dependent manner, it may be a slight effect of the test item.
No other test item-related effects were noted.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In the liver, there was centrilobular hepatocellular hypertrophy in males and females in medium- and high-dose groups. There was no further lesion along with this hypertrophy and therefore, an adaptive change in liver metabolism was concluded.
In the thyroid glands, there was diffuse follicular hypertrophy in some high dose males and females at minor degrees of severity. This was considered to be the result of increased metabolism in the liver. The changes in the liver and in the thyroid glands were not considered to be adverse.
In the kidneys of males in medium- and high-dose groups, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent alpha-2-microglobulin and the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in male rats due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.
In low-dose group, no test item-related findings were noted.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating Performance and Fertility: The mean precoital time was unaffected by treatment with the test item. Mean precoital time was 4.3, 3.0, 2.7 and 2.7 days, in order of increasing dose level. Two females in control group, and one female each in low- and high-dose groups were not pregnant.
- Duration of Gestation: The mean duration of gestation was unaffected by exposure to the test item and was similar in all dose groups.
- Corpora Lutea Count: Mean number of corpora lutea per dam (determined at necropsy) gave no indication of a test item-related effect.
- Implantation Rate and Post-implantation Loss: The mean number of implantations per dam (13.9, 14.9, 14.4, 12.7, in order of ascending dose
level) was not affected by treatment with the test item. Post-implantation loss was also unaffected by the test item (1.3, 1.1, 1.5 and 0.4, as a mean per dam and in order of increasing dose level).
- There were no effects on the completeness of stages or cell populations. There was no indication for maturation arrest, re-absorption of sperm or any other degenerative type.

Key result

Dose descriptor:

NOAEL

Remarks:

Screening for Fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related findings were noted at the first litter check or during the lactation period.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No dead pups were found at the first litter check at any dose level. Thereafter, three pups were lost from 2 litters in high-dose group. Since only 2 out of 9 litters were affected, this was considered to be incidental. No other pups were lost from any other litter.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Reduced mean body weights of the pups at 800 mg/kg bw/d, but not statistically significant and in the range of historical control data.

In high-dose group, the mean body weight of the pups was reduced on day 1 post partum (5.9 g compared to 6.4 g in the control group). In addition, the pups did not increase in weight as much as in the control group (+39.0% compared to +46.9% in the control group), resulting in reduced mean body weights of pups on day 4 post partum (8.2 g compared to 9.4 g in the control group). Since these effects were not statistically significant and in the range of the historical control data (5.7 - 6.3 g on day 1 post partum and 8.1 - 9.4 g on day 4 post partum - even though borderline to the lower limit), they were considered to be test item-related but not adverse.
The mean body weights and body weight gain of the pups in low- and medium-dose groups were not affected by treatment with the test item

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related abnormal findings were noted at necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item. The statistically significant increase in the proportion of females in high-dose group (59%) was considered to be due to biological variability.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Screening for development (foetal and pup growth survival until day 4)

Generation:

F1

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Discussion:

At 800 mg/kg bw/day, salivation was noted in isolated individuals. There was an increased incidence of a decreased number of rearings in the males in group 4 compared to the control group. In addition, body temperature was statistically significantly decreased in males and females in group 4. Although this was within the range of the historical control data, since body temperature decreased in a dose-dependent manner, it may be a slight effect of the test item. Mean food consumption and body weight gain were reduced slightly and transiently in the males and females in the first week of the pre-pairing period. Clinical laboratory investigations revealed that the level of platelets was statistically significantly increased in the males. In the females, the prothrombin time was outside the range of the historical control data. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. From the macroscopical examination, two females had an enlarged liver. Microscopically, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity. These findings were not considered to be adverse. In the kidneys of males, there was an increased degree of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent α2-microglobulin and that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

At 400 mg/kg bw/day, mean food consumption was very slightly reduced in the males during the first week of the pre-pairing period. This had no effect on the mean absolute body weights. The level of platelets was statistically significantly increased in the males. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. Microscopical examination revealed centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. The changes that were observed for platelets may be related to changed renal function and increased excretion. Biochemical data reveal also an affection of the female kidneys, by changes in protein and ion parameters. The cause of increased globulin levels remains unclear. Other biochemical changes recorded, including cholesterol and bilirubin are likely due to changes in the liver metabolism.

At 100 mg/kg bw/d, no test item-related effects were noted in the in-life phase. No effects were noted in the clinical laboratory investigations. At necropsy, no test item-related macroscopical or microscopical findings were noted.

Conclusions:

The NOAEL for fertility and development was considered to be 800 mg/kg bw/day.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/development toxicity screening test performed in accordance with OECD test guideline No. 422 and in compliance with GLP, ST 10 C 08 diluted in corn oil was administered to 10 HanRcc: WIST(SPF) rats/sex/dose by gavage at dose levels of 0, 100, 400 and 800 mg/kg bw/day. Control rats were given the vehicle alone. The test material was administered to male rats for at least 28 days and to female rats for 16 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following results were obtained for the Parent animals:

- Mortality and General tolerability

All males and females in medium- and high dose groups pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. One male and one female in high-dose group had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. These findings were considered to be a sign of discomfort following the treatment.

- Functional Observational Battery

Body temperature was statistically significantly decreased in high-dose group in males and females as well as in the females in medium-dose group. Body temperature decreased in a dose-dependent manner. However, since these changes were within the range of the historical control data, they were considered of no toxicological importance.

There was an increased incidence of a reduced number of rearings in high-dose group compared to the control group. However, in general, when only one FOB measurement is affected, the results are not considered evidence of a neurotoxic effect, mostly when the effects occurred at the high dose, in the presence of sign of toxicity such as body weigh or body temperature descreases (Guidelines for Neurotoxicity Risk Assessment, EPA, April 1998).

- Food consumption

In the males, mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period. In the females, it was statistically significantly reduced in high-dose group over the first week of the pre-pairing period. These variations are normal responses to the taste of fragrances, like ST 10 C 08.

- Body weights

In high-dose group in the males, body weight gain was statistically significantly reduced on isolated days during the pre-pairing period and on the last day of the after pairing period. In the females, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight was occasionally statistically significantly reduced. These findings were correlated to lower mean food consumption and were fully reversible; therefore they are not considered as toxicologically significant.

- Clinical Laboratory Investigations

Haematology: The level of platelets was statistically significantly increased in the males in medium- and high-dose groups (+23.2 and +33.8%, respectively) and was outside the historical control data in high-dose group. In the females in high-dose group, although the prothrombin time was not statistically significantly increased, the level was outside the range of the historical control data.

Clinical biochemistry: In both males and females, the levels of cholesterol and globulin in medium- and high-dose groups were outside the range of the historical control data. The level of protein in the males in the same groups was outside the range of the historical control data.

These changes were considered as adaptive in nature, and of no toxicological concern.

- Organ Weights

The weights and ratios of the liver were statistically significantly increased in males and females in medium- and high-dose groups.

- Macroscopical Findings and Histopathological Examinations

At macroscopical examination, two females in high-dose group were noted to have an enlarged liver.

In this dose group, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity.

These findings were considered to be adaptive responses to the test material and not to be adverse. In the kidneys of males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions representα2-microglobulin. It may be considered that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

In medium-dose group, there was centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia.

 

The following results were obtained for the Pups:

-      Findings at First Litter Check and during Lactation

No test item-related findings were noted at the first litter check or during the lactation period. Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item.

-      Pup Weights to Day 4 Post Partum

In high-dose group, the mean body weight of the pups was reduced on day 1 post partum. In addition, the pups did not increase in weight as much as in the control group, resulting in reduced mean body weights of pups on day 4 post partum. Since these effects were not statistically significant and in the range of the historical control data, they were considered to be test item-related but not adverse.

-      Macroscopical Findings

No test item-related abnormal findings were noted at necropsy.

 

Based on the results, the NOAEL for fertility and development (foetal and pup growth until Day 4) was considered to be 800 mg/kg bw/d.

 

Under the test conditions, ST 10 C 08 is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP).

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across approach is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological, ecotoxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances (Table 1) are Cycloalkane ethers. The source substance is the racemic form, while the target substance is the (-)-isomer.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is expected to have similar toxicological profile than the target substance considering the respective physico-chemical and toxicological properties.
The study design (OECD 421, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the screening for reproduction/developmental toxicity conducted in the rat with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.7.1.

4. DATA MATRIX
See Iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All males and females treated with 400 or 800 mg/kg bw/d pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. This was considered to be a sign of discomfort following treatment rather than a toxic effect of the test item. One male and one female treated with 800 mg/kg bw/d had salivation for isolated days. In addition, one female had salivation for most of the prepairing, pairing and gestation periods. This was considered to be a sign of discomfort following the treatment.
Other clinical signs noted were a wound on the shoulder of one male in low-dose group and hair loss in one dam in the control group. These findings were considered to be incidental.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Normal responses to the taste of fragrances.

- Males: In high-dose group, body weight gain was statistically significantly reduced on days 3, 6 and 10 of the pre-pairing period and on the last day of the after pairing period. However, mean body weight was similar in all dose groups throughout the study. No test item-related effects were noted in the other groups.
- Females: In high-dose group 4, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight on days 3, 4 and 6 during the pre-pairing period.This was considered to be a result of treatment with the test item. No other test item-related effects were noted during the study at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Normal responses to the taste of fragrances.

- Males: Mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period (-5.6% and -6.1%, respectively). Thereafter, food consumption was not affected by treatment with the test item in any dose group.
- Females: In high-dose group, mean food consumption was statistically significantly reduced over the first week of the pre-pairing period (-18.4% compared to the control group). This reduction was considered to be a test item-related effect. The statistically significant increase in low-dose group in the first week of the pre-pairing period was considered to be incidental due to the lack of a dose-dependent pattern.
No other test item-related effects were noted in the food consumption during the study at any dose level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Males The level of platelets was statistically significantly increased in medium-dose groups (+23.2%, compared to the control group) and 4 (+33.8%) and was outside the historical control data in high-dose group.
- Females: In the females in high-dose group, although the prothrombin time was not statistically significantly increased (+15.3%), the level was outside the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Males The level of cholesterol was statistically significantly increased in medium- and high-dose dose groups (+28.5% and +44.0%, respectively). Both these values were outside the range of the historical control data. The level of protein (+5.3% in medium-dose group and +8.6% in high-dose group) as well as globulin (+9.6% in medium-dose group and +16.5% in high-dose group) were increased and although the values were not statistically significant, they were outside the range of the historical control data.
- Females: The level of cholesterol in medium- and high-dose groups (+59.0% and +125.5%, respectively) was outside the range of the historical control data and was statistically significantly increased in high-dose group. The level of globulin was statistically significantly increased in medium- and high-dose groups (+17.3% and +16.1%, respectively) and both levels were outside the range of the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In high-dose group, there was an increased incidence in males of a reduced number of rearings (80%) compared to the control group (20%). In addition, body temperature was statistically significantly decreased in males (37.9 °C compared to the control group 38.5 °C) and females (38.3 °C compared to 39.0 °C in the control group).
In medium-dose group, the body temperature of the females was statistically significantly reduced (38.5 °C compared to 39.0 °C in the control group).
Although the body temperatures were within the range of the historical control data, since they decreased in a dose-dependent manner, it may be a slight effect of the test item.
No other test item-related effects were noted.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In the liver, there was centrilobular hepatocellular hypertrophy in males and females in medium- and high-dose groups. There was no further lesion along with this hypertrophy and therefore, an adaptive change in liver metabolism was concluded.
In the thyroid glands, there was diffuse follicular hypertrophy in some high dose males and females at minor degrees of severity. This was considered to be the result of increased metabolism in the liver. The changes in the liver and in the thyroid glands were not considered to be adverse.
In the kidneys of males in medium- and high-dose groups, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent alpha-2-microglobulin and the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in male rats due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.
In low-dose group, no test item-related findings were noted.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating Performance and Fertility: The mean precoital time was unaffected by treatment with the test item. Mean precoital time was 4.3, 3.0, 2.7 and 2.7 days, in order of increasing dose level. Two females in control group, and one female each in low- and high-dose groups were not pregnant.
- Duration of Gestation: The mean duration of gestation was unaffected by exposure to the test item and was similar in all dose groups.
- Corpora Lutea Count: Mean number of corpora lutea per dam (determined at necropsy) gave no indication of a test item-related effect.
- Implantation Rate and Post-implantation Loss: The mean number of implantations per dam (13.9, 14.9, 14.4, 12.7, in order of ascending dose
level) was not affected by treatment with the test item. Post-implantation loss was also unaffected by the test item (1.3, 1.1, 1.5 and 0.4, as a mean per dam and in order of increasing dose level).
- There were no effects on the completeness of stages or cell populations. There was no indication for maturation arrest, re-absorption of sperm or any other degenerative type.

Key result

Dose descriptor:

NOAEL

Remarks:

Screening for Fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related findings were noted at the first litter check or during the lactation period.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No dead pups were found at the first litter check at any dose level. Thereafter, three pups were lost from 2 litters in high-dose group. Since only 2 out of 9 litters were affected, this was considered to be incidental. No other pups were lost from any other litter.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Reduced mean body weights of the pups at 800 mg/kg bw/d, but not statistically significant and in the range of historical control data.

In high-dose group, the mean body weight of the pups was reduced on day 1 post partum (5.9 g compared to 6.4 g in the control group). In addition, the pups did not increase in weight as much as in the control group (+39.0% compared to +46.9% in the control group), resulting in reduced mean body weights of pups on day 4 post partum (8.2 g compared to 9.4 g in the control group). Since these effects were not statistically significant and in the range of the historical control data (5.7 - 6.3 g on day 1 post partum and 8.1 - 9.4 g on day 4 post partum - even though borderline to the lower limit), they were considered to be test item-related but not adverse.
The mean body weights and body weight gain of the pups in low- and medium-dose groups were not affected by treatment with the test item

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related abnormal findings were noted at necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item. The statistically significant increase in the proportion of females in high-dose group (59%) was considered to be due to biological variability.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Screening for development (foetal and pup growth survival until day 4)

Generation:

F1

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Discussion:

At 800 mg/kg bw/day, salivation was noted in isolated individuals. There was an increased incidence of a decreased number of rearings in the males in group 4 compared to the control group. In addition, body temperature was statistically significantly decreased in males and females in group 4. Although this was within the range of the historical control data, since body temperature decreased in a dose-dependent manner, it may be a slight effect of the test item. Mean food consumption and body weight gain were reduced slightly and transiently in the males and females in the first week of the pre-pairing period. Clinical laboratory investigations revealed that the level of platelets was statistically significantly increased in the males. In the females, the prothrombin time was outside the range of the historical control data. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. From the macroscopical examination, two females had an enlarged liver. Microscopically, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity. These findings were not considered to be adverse. In the kidneys of males, there was an increased degree of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent α2-microglobulin and that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

At 400 mg/kg bw/day, mean food consumption was very slightly reduced in the males during the first week of the pre-pairing period. This had no effect on the mean absolute body weights. The level of platelets was statistically significantly increased in the males. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. Microscopical examination revealed centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. The changes that were observed for platelets may be related to changed renal function and increased excretion. Biochemical data reveal also an affection of the female kidneys, by changes in protein and ion parameters. The cause of increased globulin levels remains unclear. Other biochemical changes recorded, including cholesterol and bilirubin are likely due to changes in the liver metabolism.

At 100 mg/kg bw/d, no test item-related effects were noted in the in-life phase. No effects were noted in the clinical laboratory investigations. At necropsy, no test item-related macroscopical or microscopical findings were noted.

Conclusions:

Based on the results, the NOAEL for the source substance for fertility and development (foetal and pup growth until Day 4) was considered to be 800 mg/kg bw/d. The target substance has the same molecular weight than the source substance so no correction is needed for this NOAEL which is therefore set to 800 mg/kg bw/day for the target substance.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/development toxicity screening test performed in accordance with OECD test guideline No. 422 and in compliance with GLP, the source substance ST 10 C 08 diluted in corn oil was administered to 10 HanRcc: WIST(SPF) rats/sex/dose by gavage at dose levels of 0, 100, 400 and 800 mg/kg bw/day. Control rats were given the vehicle alone. The test material was administered to male rats for at least 28 days and to female rats for 16 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following results were obtained for the Parent animals:

- Mortality and General tolerability

All males and females in medium- and high dose groups pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. One male and one female in high-dose group had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. These findings were considered to be a sign of discomfort following the treatment.

- Functional Observational Battery

Body temperature was statistically significantly decreased in high-dose group in males and females as well as in the females in medium-dose group. Body temperature decreased in a dose-dependent manner. However, since these changes were within the range of the historical control data, they were considered of no toxicological importance.

There was an increased incidence of a reduced number of rearings in high-dose group compared to the control group. However, in general, when only one FOB measurement is affected, the results are not considered evidence of a neurotoxic effect, mostly when the effects occurred at the high dose, in the presence of sign of toxicity such as body weigh or body temperature descreases (Guidelines for Neurotoxicity Risk Assessment, EPA, April 1998).

- Food consumption

In the males, mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period. In the females, it was statistically significantly reduced in high-dose group over the first week of the pre-pairing period. These variations are normal responses to the taste of fragrances, like ST 10 C 08.

- Body weights

In high-dose group in the males, body weight gain was statistically significantly reduced on isolated days during the pre-pairing period and on the last day of the after pairing period. In the females, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight was occasionally statistically significantly reduced. These findings were correlated to lower mean food consumption and were fully reversible; therefore they are not considered as toxicologically significant.

- Clinical Laboratory Investigations

Haematology: The level of platelets was statistically significantly increased in the males in medium- and high-dose groups (+23.2 and +33.8%, respectively) and was outside the historical control data in high-dose group. In the females in high-dose group, although the prothrombin time was not statistically significantly increased, the level was outside the range of the historical control data.

Clinical biochemistry: In both males and females, the levels of cholesterol and globulin in medium- and high-dose groups were outside the range of the historical control data. The level of protein in the males in the same groups was outside the range of the historical control data.

These changes were considered as adaptive in nature, and of no toxicological concern.

- Organ Weights

The weights and ratios of the liver were statistically significantly increased in males and females in medium- and high-dose groups.

- Macroscopical Findings and Histopathological Examinations

At macroscopical examination, two females in high-dose group were noted to have an enlarged liver.

In this dose group, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity.

These findings were considered to be adaptive responses to the test material and not to be adverse. In the kidneys of males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions representα2-microglobulin. It may be considered that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

In medium-dose group, there was centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia.

 

The following results were obtained for the Pups:

-      Findings at First Litter Check and during Lactation

No test item-related findings were noted at the first litter check or during the lactation period. Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item.

-      Pup Weights to Day 4 Post Partum

In high-dose group, the mean body weight of the pups was reduced on day 1 post partum. In addition, the pups did not increase in weight as much as in the control group, resulting in reduced mean body weights of pups on day 4 post partum. Since these effects were not statistically significant and in the range of the historical control data, they were considered to be test item-related but not adverse.

-      Macroscopical Findings

No test item-related abnormal findings were noted at necropsy.

 

Based on the results, the NOAEL for the source substance for fertility and development (foetal and pup growth until Day 4) was considered to be 800 mg/kg bw/d. The target substance has the same molecular weight than the source substance so no correction is needed for this NOAEL which is therefore set to 800 mg/kg bw/day for the target substance.

 

Under the test conditions, ST 10 C 08 is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No study was available on the target substance, therefore a read-across approach was used. The source substance is considered adequate for read-across purposes as data relates to the racemic form of the registered substance (see IUCLID section 13 for additional justification).

 

In the dose range-finding study for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Harlan, 2009, rel.2), 3 HanRcc : WIST(SPF) rats/sex/dose were treated by gavage at the dose levels of 0, 100, 300 or 1000 mg ST 10 C 08/kg bw/d. Males were treated over a 14-day pre-pairing period and during the pairing period and up to one day before necropsy (for a total of at least 28 days of treatment). Females were treated throughout the pre-pairing, pairing and gestation period up to day 13 post coitum. At the high dose level, reduced food consumption and body weight gain were noticed but the main finding was that pre-implantation loss was increased and the number of implantation sites was decreased. However, the data showed that of the two pregnant females at the maximum dose level, one had normal data and the other had only one implant. Due to the small number of females used in this range-finding study, it is difficult to estimate if this observation is related to treatment or due to an abnormal female. Using a worst-case approach, the high-dose level to be used in the main study was lowered to 800 mg/kg bw/d.

 

In the main study, considered as the key study (Harlan, 2009, rel. 2), the test material diluted in corn oil was administered orally, by gavage, at dose levels of 0, 100, 400 or 800 mg/kg bw/d, to rats according to the OECD test guideline No. 422 and in compliance with GLP.

No deaths occurred after the administration of the test material. Some effects of discomfort, such as salivation, were observed following treatment at the two highest dose levels. Increased liver weight, biochemical changes (bilirubin, cholesterol, protein) and liver hypertrophy were considered to be part of an adaptive response to an increase in metabolic demand. There were no reproductive or developmental effects at any dose level. Therefore, the concern raised by the data of one animal in the range-finder was alleviated.

The No Observed Adverse Effect Level (NOAEL) for fertility is therefore 800 mg/kg bw/d. The target substance has the same molecular weight than the source substance so no correction is needed for this NOAEL which is therefore set to 800 mg/kg bw/day for the target substance.

 

As no alerts for reproductive toxicity were provided in the main study, no additional study is proposed at this tonnage level.

Effects on developmental toxicity

Description of key information

Read-across on racemic form of Cycloalkane ethers:

• Prenatal developmental toxicity study in rats (OECD 414, GLP, rel.1, Key study): maternal NOAEL = 400 mg/kg bw/day; embryo-fetal NOAEL = 400 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to IUCLID section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across approach is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological, ecotoxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances (Table 1) are Cycloalkane ethers. The source substance is the racemic form, while the target substance is the (-)-isomer.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is expected to have similar toxicological profile than the target substance considering the respective physico-chemical and toxicological properties.
The study design (OECD 414, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the developmental toxicity/teratogenicity study conducted in the rat with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex IX, 8.7.2.

4. DATA MATRIX
See IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At routine physical examination three females receiving 800 mg/kg/day were considered to have a thin build; there were no other signs that were considered to relate to treatment.
At 400 or 800 mg/kg/day an increased incidence of piloerection was observed in association with dose administration.
In addition, chin rubbing and increased salivation was observed for a few animals receiving 400 or 800 mg/kg/day; this is commonly observed in oral gavage studies and is considered to relate to palatability of the formulation, rather than a direct effect of the test item and is therefore considered to be of no toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (no. 74) receiving 800 mg/kg/day was killed for welfare reasons on GD7 due to a swollen, dark hindlimb and loss of locomotion. Macroscopic examination revealed tibiotarsal trauma. This death was accidental and unrelated to administration of ST 10 C 08.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute body weights were statistically significantly low when compared with Controls for females receiving 800 mg/kg/day from GD8 to GD10, and on GD19 and GD20. Overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From GD6 to GD9 females receiving 800 mg/kg/day showed low consumption when compared with Controls (p<0.01); subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of alightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum T3 and T4 concentrations in animals treated with ST 10 C 08 were not statistically significant different from mean concurrent Control values.
Administration by oral gavage of ST 10 C 08 at 800 mg/kg/day had a statistically significant effect on serum TSH levels in female rats when compared to the concurrent control group (p<0.01); circulating levels at 100 or 400 mg/kg/day were similar to Controls.

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

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):

Animals that received 400 or 800 mg/kg/day showed high mean thyroid weight at approximately 125% (p<0.05) and 142 % (p<0.01) of Controls, respectively. this correlated with the microscopic finding of follicular hypertrophy in some animals.
The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01). The maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At terminal sacrifice, thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day ST 10 C 08 and in one female administered 400 mg/kg/day ST 10 C 08.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

See table of results in the section "Attached background material".

Number of abortions:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on the mean number of implantations or sex ratio at any dose level investigated.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of early resorptions were slightly high at 800 mg/kg/day and when compared with Controls the resultant post-implantation loss was high (p<0.05) and the live litter size was low (p<0.05). At 100 or 400 mg/kg/day the mean number of resorptions, the extent of the pre- and post- implantation loss and total number of live young were similar Controls and considered to be unaffected by treatment.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early resorptions were slightly high at 800 mg/kg/day and when compared with Controls the resultant post-implantation loss was high (p<0.05) and the live litter size was low (p<0.05). At 100 or 400 mg/kg/day the mean number of resorptions, the extent of the pre- and post- implantation loss and total number of live young were similar Controls and considered to be unaffected by treatment.

Dead fetuses:

no effects observed

Description (incidence and severity):

At 100 or 400 mg/kg/day the total number of live young were similar Controls and considered to be unaffected by treatment.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 20 after mating, one female (no. 48) that received 400 mg/kg/day and one female (no. 79) that received 800 mg/kg/day were not pregnant. Macroscopic examination of female no. 48 revealed dark fluid in the uterine cervix, marked fluid distention of the uterus, and a blind portion of the vagina; female no. 79 showed no abnormality. All remaining animals were pregnant.

Details on maternal toxic effects:

Oral administration of ST 10 C 08 to pregnant Han Wistar rats during organogenesis and the fetal growth phase at 100, 400 and 800 mg/kg/day was generally well tolerated by maternal animals in life with effects on general condition limited to thin build for three animals at 800 mg/kg/day. There were no adverse effects on either maternal body weight gain, food consumption, thyroid hormones or macropathology.

Maternal thyroid weight however was significantly high at 400 or 800 mg/kg/day, and this correlated with the microscopic finding of follicular hypertrophy in some animals at these dose levels and circulating levels of Thyroid Stimulating hormone (TSH) were slightly high at 800 mg/kg/day. However, in the absence of an effect on thyroid weight or serum levels of either, Triiodothyronine (T3) or Thyroxine (T4), these findings were considered not to be adverse.
The gravid uterine weight at 800 mg/kg/day was significantly low when compared with Controls and this correlated with the high post-implantation loss, resultant low litter size and low fetal weight at this dose level; these parameters were unaffected at 100 or 400 mg/kg/day.

See the table of results in the attachments section.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean placental weight was unaffected by treatment at dose levels up to and including 800 mg/kg/day. At 800 mg/kg/day the mean total litter weight was statistically significantly low when compared with Controls (p<0.01) and the mean overall fetal weight was also slightly low at 92% of Controls (p<0.05). The mean total litter weight and mean fetal weight were unaffected at 100 and 400 mg/kg/day.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The live litter size was low (p<0.05) at 800 mg/kg/day.
At 100 or 400 mg/kg/day the total number of live young were similar Controls and considered to be unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the mean total litter weight was statistically significantly low when compared with Controls (p<0.01) and the mean overall fetal weight was also slightly low at 92% of Controls (p<0.05). The mean total litter weight and mean fetal weight were unaffected at 100 and 400 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The fetal ano-genital distance was unaffected by maternal treatment.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.
At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight and are not considered adverse.
At 100 mg/kg/day one fetus in litter no. 34 had exencephaly which was within the historical control range, this fetus also had open eyelids which is considered secondary to the exencephaly. So, although the major abnormality of open eyelids exceeded HCD it is not considered to be treatment related as it is related to the exencephaly which was within HCD and was not observed at 400 or 800 mg/kg/day.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Detailed fetal examination revealed a low incidence of major predominately unrelated abnormalities at all dose levels the majority of which exceeded the historical control data range. The highest incidence of major abnormalities was seen at 800 mg/kg/day, in particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly. The increased post-implantation loss at 800 mg/kg/day and the high incidence of major abnormalities at this dose level are considered to be interrelated. At 400 mg/kg/day there were also a number of major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.
See the table of results in the attachments section.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Remarks on result:

other:

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: ear

external: eye

external: anus

skeletal: skull

visceral/soft tissue: central nervous system

visceral/soft tissue: eye

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities: Four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

Key result

Developmental effects observed:

no

Formulation analysis:

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.
The difference from mean remained within 2%, confirming precise analysis, except for first Week where Group 2 had a difference from mean value of 6.70%. As the two individual values for Group 2 were within the +10/-15% acceptance criteria and the RME value was within the +10/-15% acceptance criteria, it is considered the formulations were accurately prepared. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure.

Conclusions:

Based on the results of this study, the maternal NOAEL was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal NOAEL was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day) for the source substance. The target substance has the same molecular weight than the source substance so no correction is needed for these NOAELs which are therefore set to 400 mg/kg bw/day for maternal and embryo-fetal NOAELs respectively for the target substance.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 females received ST 10 C 08 at doses of 100, 400 or 800 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterine weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.

 

Maternal responses

Administration of ST 10 C 08 at 100, 400 or 800 mg/kg/day resulted in high TSH levels at 800 mg/kg/day but no statistically significant effect on serum T3/T4 levels in pregnant female rats; TSH levels at 100 or 400 mg/kg/day were similar to Controls.

At routine physical examination the build for three females receiving 800 mg/kg/day was considered thin.
At 800 mg/kg/day mean absolute body weights from GD8-10 and on GD19/20 were statistically significantly low when compared with Controls; the overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01) but the maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day food consumption was low from GD6 to GD9; subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of slightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Animals that received 400 or 800 mg/kg/day showed significantly high mean thyroid weight; a dose response was apparent.
No maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day and in one female at 400 mg/kg/day. This effect was concordant with effects occuring in a 90-day repeated dose toxicity study (Labcorp study No. 8449089, see reference to other study). In this study, minimal to slight follicular cell hypertrophy noted in males and females administered 400 or 800 mg/kg/day and males administered 100 mg/kg/day correlated with increased absolute and statistically significant body weight-adjusted thyroid gland weights for males. This finding is commonly encountered in rats in combination with centrilobular hypertrophy of the liver and is a consequence of hepatic microsomal enzyme induction causing increased clearance of thyroid hormones and feedback through the pituitary-thyroid axis (Curran and Degroot, 1991). Although microscopic examination of the liver was not evaluated in this study, we can assume that these thyroid follicular cell hypertrophy effects are a consequence of the induction of liver microsomal enzymes.

 

Litter responses

The mean number of early resorptions and post-implantation loss were high at 800 mg/kg/day and the resultant live litter size was low; litter data was unaffected by maternal treatment at 100 or 400 mg/kg/day.

At 800 mg/kg/day the mean total litter weight and the mean overall fetal weight were low; these parameters were unaffected at 100 or 400 mg/kg/day.

The fetal ano-genital distance was unaffected by maternal treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities.  In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. 

At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight seen at these dose levels and are not considered adverse.

 

Based on the results of this study, the maternal NOAEL was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal NOAEL was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day) for the source substance. The target substance has the same molecular weight than the source substance so no correction is needed for these NOAELs which are therefore set to 400 mg/kg bw/day for maternal and embryo-fetal NOAELs respectively for the target substance.

Based on the results of this study, the source and the target substances are not classified according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP). This study is considered as acceptable and satisfies the requirement for developmental toxicity/teratogenicity endpoint.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 March 2021 to 07 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 414 Guideline without deviations.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST

Details on test animals or test system and environmental conditions:

RATIONALE FOR ANIMAL MODEL:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar rat strain was used because of the historical control data available at this laboratory.

TEST ANIMALS
- Source: Envigo RMS Limited.
- Age at study initiation: 78 to 84 days old.
- Weight at study initiation: 169 to 219 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Six days before pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
Environmental Enrichment
- Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

IN-LIFE DATES: From: 31 March 2021 To: 30 April 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 25, 100 and 200 mg/mL
- Dose volume: 4 mL/kg

STABILITY AND HOMOGENEITY
Before commencement of treatment, the suitability of the proposed mixing procedures was determined as part of another study, Covance GLP Study 8449087. In that study formulations in the range 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Stability was determined as 15 days refrigerated (2 to 8°C) and for one day at ambient temperature (15 to 25°C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: Samples of each formulation prepared for administration in first and last week of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:

- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

From mating (Day 0) to necropsy (Day 20).

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Test item

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Test item

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Test item

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 100, 400 or 800 mg/kg/day) were selected in conjunction with the Sponsor. A combined repeat dose toxicity study with reproductive/developmental toxicity screening test (OECD 422) was conducted with the test substance in 2008/2009 (Harlan study no C05606, Füllinsdorf, Switzerland) using Han Wistar rats. In that study, the test substance was administered to rats (n = 10/group) at doses of 0, 100, 400 or 800 mg/kg/day in corn oil by oral gavage. The general NOAEL and NOEL from that study were determined to be 800 and 100 mg/kg/day, respectively. The developmental NOAEL and NOEL were determined to be 800 and 400 mg/kg/day, respectively. Based on the observations from that study it would be anticipated that animals dosed using a limit dose approach (i.e., high dose of 1000 mg/g/day) would be expected to exhibit adverse effects that would not be consistent with animal welfare testing requirements. Therefore, dose levels of 0, 100, 400, and 800 mg/kg/day were selected for use on this OECD 414 study.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Maternal examinations:

MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration: A pre-dose observation; One to two hours after completion of dosing and as late as possible in the working day.
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Animals surviving until the end of the scheduled study period were killed on Day 20 after mating by Carbon dioxide asphyxiation.
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in 10% Neutral Buffered Formalin. Terminal blood samples were also required for thyroid hormone analysis.
- Organ weights: the organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 7.8.2/1.
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
- Histology: For all adult females, tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Light microscopy: Thyroid was preserved for examination for premature deaths and terminal sacrifice of all animals. Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Ovaries and uterine content:

For females surviving to term, the ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight (including cervix and ovaries): Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of Fetuses (live and dead): Yes

In apparently non pregnant animals and for apparently empty uterine horns, the number of uterine implantation sites were checked after staining with ammonium sulphide.

Blood sampling:

THYROID HORMONE ANALYSIS: Yes
- Blood samples (1.0 mL) were collected at termination of the study in all surviving adults in sublingual vein after an isoflurane anaesthesia. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (2000 g for 10-min at 4°C).
- Number of aliquots: Two per animal. Aliquot 1: 0.2 mL serum for T3/T4; Aliquot 2: residual serum for TSH.

Fetal examinations:

FETAL EXAMINATION & PROCESSING
- Method of kill for fetuses: Chilling on a cool plate (approximately 0 °C)
- Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded and particular attention was paid to external genital organs of male fetuses. The sex and ano genital distance of each fetus was recorded.
- Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
- Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
- Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

FETAL PATHOLOGY EXAMINATION
- Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
- Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.

Statistics:

See " Any other information on materials and methods incl. tables"

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss
was considered to reflect losses due to non-fertilization of ova and failure to implant. It was
calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At routine physical examination three females receiving 800 mg/kg/day were considered to have a thin build; there were no other signs that were considered to relate to treatment.
At 400 or 800 mg/kg/day an increased incidence of piloerection was observed in association with dose administration.
In addition, chin rubbing and increased salivation was observed for a few animals receiving 400 or 800 mg/kg/day; this is commonly observed in oral gavage studies and is considered to relate to palatability of the formulation, rather than a direct effect of the test item and is therefore considered to be of no toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (no. 74) receiving 800 mg/kg/day was killed for welfare reasons on GD7 due to a swollen, dark hindlimb and loss of locomotion. Macroscopic examination revealed tibiotarsal trauma. This death was accidental and unrelated to administration of ST 10 C 08.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute body weights were statistically significantly low when compared with Controls for females receiving 800 mg/kg/day from GD8 to GD10, and on GD19 and GD20. Overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From GD6 to GD9 females receiving 800 mg/kg/day showed low consumption when compared with Controls (p<0.01); subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of alightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum T3 and T4 concentrations in animals treated with ST 10 C 08 were not statistically significant different from mean concurrent Control values.
Administration by oral gavage of ST 10 C 08 at 800 mg/kg/day had a statistically significant effect on serum TSH levels in female rats when compared to the concurrent control group (p<0.01); circulating levels at 100 or 400 mg/kg/day were similar to Controls.

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

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):

Animals that received 400 or 800 mg/kg/day showed high mean thyroid weight at approximately 125% (p<0.05) and 142 % (p<0.01) of Controls, respectively. this correlated with the microscopic finding of follicular hypertrophy in some animals.
The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01). The maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At terminal sacrifice, thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day ST 10 C 08 and in one female administered 400 mg/kg/day ST 10 C 08.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

See table of results in the section "Attached background material".

Number of abortions:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on the mean number of implantations or sex ratio at any dose level investigated.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of early resorptions were slightly high at 800 mg/kg/day and when compared with Controls the resultant post-implantation loss was high (p<0.05) and the live litter size was low (p<0.05). At 100 or 400 mg/kg/day the mean number of resorptions, the extent of the pre- and post- implantation loss and total number of live young were similar Controls and considered to be unaffected by treatment.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early resorptions were slightly high at 800 mg/kg/day and when compared with Controls the resultant post-implantation loss was high (p<0.05) and the live litter size was low (p<0.05). At 100 or 400 mg/kg/day the mean number of resorptions, the extent of the pre- and post- implantation loss and total number of live young were similar Controls and considered to be unaffected by treatment.

Dead fetuses:

no effects observed

Description (incidence and severity):

At 100 or 400 mg/kg/day the total number of live young were similar Controls and considered to be unaffected by treatment.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 20 after mating, one female (no. 48) that received 400 mg/kg/day and one female (no. 79) that received 800 mg/kg/day were not pregnant. Macroscopic examination of female no. 48 revealed dark fluid in the uterine cervix, marked fluid distention of the uterus, and a blind portion of the vagina; female no. 79 showed no abnormality. All remaining animals were pregnant.

Details on maternal toxic effects:

Oral administration of ST 10 C 08 to pregnant Han Wistar rats during organogenesis and the fetal growth phase at 100, 400 and 800 mg/kg/day was generally well tolerated by maternal animals in life with effects on general condition limited to thin build for three animals at 800 mg/kg/day. There were no adverse effects on either maternal body weight gain, food consumption, thyroid hormones or macropathology.

Maternal thyroid weight however was significantly high at 400 or 800 mg/kg/day, and this correlated with the microscopic finding of follicular hypertrophy in some animals at these dose levels and circulating levels of Thyroid Stimulating hormone (TSH) were slightly high at 800 mg/kg/day. However, in the absence of an effect on thyroid weight or serum levels of either, Triiodothyronine (T3) or Thyroxine (T4), these findings were considered not to be adverse.
The gravid uterine weight at 800 mg/kg/day was significantly low when compared with Controls and this correlated with the high post-implantation loss, resultant low litter size and low fetal weight at this dose level; these parameters were unaffected at 100 or 400 mg/kg/day.

See the table of results in the attachments section.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean placental weight was unaffected by treatment at dose levels up to and including 800 mg/kg/day. At 800 mg/kg/day the mean total litter weight was statistically significantly low when compared with Controls (p<0.01) and the mean overall fetal weight was also slightly low at 92% of Controls (p<0.05). The mean total litter weight and mean fetal weight were unaffected at 100 and 400 mg/kg/day.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The live litter size was low (p<0.05) at 800 mg/kg/day.
At 100 or 400 mg/kg/day the total number of live young were similar Controls and considered to be unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the mean total litter weight was statistically significantly low when compared with Controls (p<0.01) and the mean overall fetal weight was also slightly low at 92% of Controls (p<0.05). The mean total litter weight and mean fetal weight were unaffected at 100 and 400 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The fetal ano-genital distance was unaffected by maternal treatment.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.
At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight and are not considered adverse.
At 100 mg/kg/day one fetus in litter no. 34 had exencephaly which was within the historical control range, this fetus also had open eyelids which is considered secondary to the exencephaly. So, although the major abnormality of open eyelids exceeded HCD it is not considered to be treatment related as it is related to the exencephaly which was within HCD and was not observed at 400 or 800 mg/kg/day.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities. In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, with the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range.
At 400 mg/kg/day there were a number of unrelated major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Detailed fetal examination revealed a low incidence of major predominately unrelated abnormalities at all dose levels the majority of which exceeded the historical control data range. The highest incidence of major abnormalities was seen at 800 mg/kg/day, in particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly. The increased post-implantation loss at 800 mg/kg/day and the high incidence of major abnormalities at this dose level are considered to be interrelated. At 400 mg/kg/day there were also a number of major findings, at low incidence and the following were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury, imperforate anus.
See the table of results in the attachments section.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Remarks on result:

other:

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: ear

external: eye

external: anus

skeletal: skull

visceral/soft tissue: central nervous system

visceral/soft tissue: eye

Description (incidence and severity):

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities: Four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

Key result

Developmental effects observed:

no

Formulation analysis:

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.
The difference from mean remained within 2%, confirming precise analysis, except for first Week where Group 2 had a difference from mean value of 6.70%. As the two individual values for Group 2 were within the +10/-15% acceptance criteria and the RME value was within the +10/-15% acceptance criteria, it is considered the formulations were accurately prepared. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure.

Conclusions:

Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day).

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 females received ST 10 C 08 at doses of 100, 400 or 800 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterine weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.

 

Maternal responses

Administration of ST 10 C 08 at 100, 400 or 800 mg/kg/day resulted in high TSH levels at 800 mg/kg/day but no statistically significant effect on serum T3/T4 levels in pregnant female rats; TSH levels at 100 or 400 mg/kg/day were similar to Controls.

At routine physical examination the build for three females receiving 800 mg/kg/day was considered thin.
At 800 mg/kg/day mean absolute body weights from GD8-10 and on GD19/20 were statistically significantly low when compared with Controls; the overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01) but the maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day food consumption was low from GD6 to GD9; subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of slightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Animals that received 400 or 800 mg/kg/day showed significantly high mean thyroid weight; a dose response was apparent.
No maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day and in one female at 400 mg/kg/day. This effect was concordant with effects occuring in a 90-day repeated dose toxicity study (Labcorp study No. 8449089, see reference to other study). In this study, minimal to slight follicular cell hypertrophy noted in males and females administered 400 or 800 mg/kg/day and males administered 100 mg/kg/day correlated with increased absolute and statistically significant body weight-adjusted thyroid gland weights for males. This finding is commonly encountered in rats in combination with centrilobular hypertrophy of the liver and is a consequence of hepatic microsomal enzyme induction causing increased clearance of thyroid hormones and feedback through the pituitary-thyroid axis (Curran and Degroot, 1991). Although microscopic examination of the liver was not evaluated in this study, we can assume that these thyroid follicular cell hypertrophy effects are a consequence of the induction of liver microsomal enzymes.

 

Litter responses

The mean number of early resorptions and post-implantation loss were high at 800 mg/kg/day and the resultant live litter size was low; litter data was unaffected by maternal treatment at 100 or 400 mg/kg/day.

At 800 mg/kg/day the mean total litter weight and the mean overall fetal weight were low; these parameters were unaffected at 100 or 400 mg/kg/day.

The fetal ano-genital distance was unaffected by maternal treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities.  In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. 

At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight seen at these dose levels and are not considered adverse.

 

Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study used for endpoint conclusion is GLP-compliant and of high quality. Additional developmental toxicity studies are not deemed necessary.

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

No study was available on the target substance, therefore a read-across approach was used. The source substance is considered adequate for read-across purposes as data relates to the racemic form of the registered substance (see IUCLID section 13 for additional justification).

 

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 females received ST 10 C 08 at doses of 100, 400 or 800 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterine weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

The mean concentrations of formulations taken during the course of the study were within 4% of the nominal concentration, confirming the accuracy of formulation.

 

Maternal responses

Administration of ST 10 C 08 at 100, 400 or 800 mg/kg/day resulted in high TSH levels at 800 mg/kg/day but no statistically significant effect on serum T3/T4 levels in pregnant female rats; TSH levels at 100 or 400 mg/kg/day were similar to Controls.

At routine physical examination the build for three females receiving 800 mg/kg/day was considered thin.
At 800 mg/kg/day mean absolute body weights from GD8-10 and on GD19/20 were statistically significantly low when compared with Controls; the overall body weight gain at 800 mg/kg/day was low at approximately 84% of Controls; however, this difference did not attain statistical significance. Body weight gain at 100 or 400 mg/kg/day was unaffected by treatment.

The gravid uterine weight was statistically significantly low when compared with Controls for females that received 800 mg/kg/day (p<0.01) but the maternal body weight gain, following adjustment for the gravid uterine weight, showed no adverse effect of treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day food consumption was low from GD6 to GD9; subsequent consumption was similar or slightly higher than Controls. At 400 mg/kg/day there were periods of slightly high consumption late gestation, but overall food consumption was unaffected by treatment.

Animals that received 400 or 800 mg/kg/day showed significantly high mean thyroid weight; a dose response was apparent.
No maternal macroscopic abnormalities detected at scheduled termination that were considered to relate to administration of ST 10 C 08.

Thyroid follicular cell hypertrophy (minimal severity) was seen in eight females administered 800 mg/kg/day and in one female at 400 mg/kg/day. This effect was concordant with effects occuring in a 90-day repeated dose toxicity study (Labcorp study No. 8449089, see reference to other study). In this study, minimal to slight follicular cell hypertrophy noted in males and females administered 400 or 800 mg/kg/day and males administered 100 mg/kg/day correlated with increased absolute and statistically significant body weight-adjusted thyroid gland weights for males. This finding is commonly encountered in rats in combination with centrilobular hypertrophy of the liver and is a consequence of hepatic microsomal enzyme induction causing increased clearance of thyroid hormones and feedback through the pituitary-thyroid axis (Curran and Degroot, 1991). Although microscopic examination of the liver was not evaluated in this study, we can assume that these thyroid follicular cell hypertrophy effects are a consequence of the induction of liver microsomal enzymes.

 

Litter responses

The mean number of early resorptions and post-implantation loss were high at 800 mg/kg/day and the resultant live litter size was low; litter data was unaffected by maternal treatment at 100 or 400 mg/kg/day.

At 800 mg/kg/day the mean total litter weight and the mean overall fetal weight were low; these parameters were unaffected at 100 or 400 mg/kg/day.

The fetal ano-genital distance was unaffected by maternal treatment at dose levels up to and including 800 mg/kg/day.

At 800 mg/kg/day the incidence of major abnormalities was high, including a number of skeletal and visceral abnormalities.  In particular four fetuses from three litters exhibited Anophthalmia/Microphthalmia/Hydroceophaly, the incidence for Hydrocephaly and Microphthalmia exceeding the historical control range. 

At 400 mg/kg/day there were also a number of unrelated major findings, at low incidence but a number were outside of the historical control data (HCD) range: absent tympanic annula, Agnathia, absent orbital socket(s), Microcephaly, Anury and imperforate anus.

At 400 and 800mg/kg/day there was a slight increase in incidence of shiny skin, 20 thoracolumbar vertebrae, incompletely ossified cranial centres and caudal vertebrae when compared with concurrent control and outside of HCD range (with the exception of cranial centres). These findings are indicative of fetal immaturity and are often associated with low mean fetal weight seen at these dose levels and are not considered adverse.

 

Based on the results of this study, the maternal NOAEL was 400 mg/kg bw/day (based on high post-implantation loss and low gravid uterine weight at 800 mg/kg/day) and the embryo-fetal NOAEL was 400 mg/kg bw/day (based on major squeletal and visceral abnormalities at 800 mg/kg bw/day) for the source substance. The target substance has the same molecular weight than the source substance so no correction is needed for these NOAELs which are therefore set to 400 mg/kg bw/day for maternal and embryo-fetal NOAELs respectively for the target substance.

Based on the results of this study, the source and the target substances are not classified according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP). This study is considered as acceptable and satisfies the requirement for developmental toxicity/teratogenicity endpoint.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data on a supporting substance, no additional classification is proposed according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP).