Tetrahydromethylphthalic anhydride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined repeated dose toxicity/reproduction and developmental toxicity study with rats according to OECD 422, the NOAEL for the test item is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring.

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-12-06 to 1997-06-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (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)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river Japan
- Age at study initiation: purchase at 9 weeks old, 1st. administration at 10 weeks old
- Weight at study initiation: male: 356.3-394.4 g, female: 213.5-252.9 g
- Fasting period before study: 18 hr
- Housing: dosing period: stainless hanger gage, one animal/gage, mating period: polycarbonate gage with wood chip
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26
- Humidity (%): 45-65
- Air changes (per hr): 13 times/hr
- Photoperiod (hrs dark / hrs light): 12/12 AM07:00-PM07:00

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle: Corn oil is used generally
- Concentration in vehicle: 0.6, 2 and 6 w/v%
- Amount of vehicle: 5 mL/kg
- Lot/batch no.: V5P5831, nakalai tesque Co.

Details on mating procedure:

Male/female per cage: 1/1,
length of cohabitation: maximal 14 days, until proof of pregnancy (formation of vaginal closing or sperm detection in vagina)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no details given

Duration of treatment / exposure:

Males; for 49 days Females; from 14 days before mating to day 3 of lactation (38 days in total)

Frequency of treatment:

one administration/day

Details on study schedule:

no details given

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Doses is 0, 30, 100 and 300 mg/kg. 12 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The doses were based on the results of a preliminary study. Repeated administration of 100, 300 and 1000 mg/kg bw/d of the test substance for 2 weeks caused the death of one male and one female in the 1000 mg/kg bw/d group. In this group there was also a tendency to suppressed body weight gain, decreased food intake, increased white blood cell count, decreased red blood cells, decreased total protein and albumin, thickening and white spots in the forestomach mucosa, and increased adrenal gland. Thickening of the forestomach mucosa was also observed in the 300 mg/kg bw/d group. No effects were observed in the 100 mg/kg bw/d group. Therefore, the high dose in the present study was set at 300 mg/kg bw/d, which is approximately 1/3 of the 1000 mg/kg bw/d dose at which excessive toxicity (i.e. death) were observed. The intermediate and low doses were set to 100 and 30 mg/kg bw/d, respectively.

Positive control:

no details given

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: one time per day
- Cage side observations: general symptom

DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: male: two times/week
female: two times/ week at pre-mating period, in pregnant period: day at 0, 4, 7,10, 14, 17 and 21, in lactation period: the day 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
male: two times/week
female: two times/ week at pre-mating period, in pregnant period: day at 1, 4, 7,10, 14, 17 and 21, in lactation period: the day 1 and 4.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after dosing period
- Anaesthetic used for blood collection: Yes (identity): Pentobarbital-Na i.p.
- Animals fasted: Yes
- How many animals: All of male
- Parameters checked in table (see below in remarks field) were examined.: WBC, RBC, Hb, Ht, PLT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after dosing period (same time for HAEMATOLOGY)
- Animals fasted: Yes
- How many animals:All of male
- Parameters checked in table were examined.: TP, ALB, A/G, Bil, GOT, GPT, TGace, ALP, TG, PL, Giu, BUN, CRE, P, Ca, Na, K, Cl

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Oestrous cyclicity (parental animals):

no details given

Sperm parameters (parental animals):

no details given

Litter observations:

Body weight (at day of birth and day 4 after birth), sex, surface abnormality at day of birth.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals, after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
organ weight: brain, heart, lung, thymus, liver, spleen, kidney, adrenal, testis, epididymis, ovary.
microscopic investigation: all animals in control, 300 mg/kg group; brain, pituitary gland, eyeball, thyroid gland, parathyroid gland, thymus, heart, lung, liver, kidney, adrenal, spleen, stomach, small intestine, large intestine, pancreas, urinary bladder, bone marrow, ovary, uterus, vagina, mammary gland.
Unfertilized animals in any groups: testes, epididymis and ovary

Postmortem examinations (offspring):

Full macroscopic examinations on all of pups Parameters assessed during study.

Statistics:

bartlett method,
standard variance: Dunnett, multiple comparison
non-standard variance: Steel, multiple comparison

Reproductive indices:

No. of pairs with successful copulation
Copulation index (No. of pairs with successful copulation/No. of pairs mated x 100)
Pairing days until copulation
No. of pregnant females
Fertility index = (No. of pregnant animals x 100/No. of pairs with successful copulation),
No. of corpora lutea
No. of implantation sites
No. of living pregnant females
No. of pregnant females with parturition gestation length
No. of pregnant females with live pups on day 0
Gestation index (No. of females with live pups x 100/No. of living pregnant females)
Delivery index (No. of pups born x 100/No. of implantation sites)

Offspring viability indices:

No. of pups alive on day 0 of lactation
Live birth index (No. of live pups on day 0 x 100/No. of pups born)
Sex ratio (Total No. of male pups/Total No. of female pups)
No. of pups alive on day 4 of lactation, body wt. of live pups (on day 0 and 4)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was seen in males treated at 300 mg/kg bw/d starting at treatment day 36. This finding is considered as non-adverse as it occurred only sporadically in some males.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three deaths were obeserved due to administraton errors (1 male and 1 female at 300 mg/kg bw/d, 1 female at 30 mg/kg be/d).

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Please refer to IUCLID section 7.5.1 (repeated dose toxicity oral).

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please refer to IUCLID section 7.5.1 (repeated dose toxicity oral).

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

For details on results for parental animals, please refer to IUCLID section 7.5.1 (repeated dose toxicity oral).
Reproduction parameters:
- At 30 and 100 mg/kg, there was a tendency for decrease of estrous frequency, but at 300 mg/kg, no statistically significant effects were observed.
- For details see tables below.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverses systemic effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No adverse effects were reported.
- At 30 and 100 mg/kg, statistically significant decrease of birth index was observed, and at 300 mg/kg, stillborn was observed only one animal (not statistically significant).
- At 100 mg/kg, total litter loss in two dams were observed.
- At 300 mg/kg, no statistically significant effects were observed.

- For details see attached documents.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects were observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Reproduction parameters

Dose level (mg/kg/day)	0	30	100	300
No. of dams	11	11	11	10
No. of corpora lutea (Mean +/- SD)	22.18 +/-0.40	22.27 +/-0.47	22.09 +/-0.30	22.30  +/-0.48
No. of implantations (Mean +/- SD)	183 16.64 +/- 1.63	188 17.09 +/- 1.30	188 17.09 +/- 0.94	162 16.20 +/- 1.03
No. of litter (Mean +/- SD)	171 15.55 +/- 2.58	178 16.18 +/- 1.25	181 16.45 +/- 0.69	154 15.40 +/- 1.65
Gestation Index	100	100	100	100
No. of stillborns Male Female Total %	0 0 0 (0)	4 4 8 (5.06)*	3 6 9 (5.33)*	4 6 10 (6.76)
No. of live newborns (Mean +/- SD)	162 14.73 +/- 2.65	150 13.64 +/- 1.43	160 14.55 +/- 0.82	138 13.80 +/- 2.53
Birth index	94.74	84.27*	88.40*	89.61
Sex ratio of live newborns (male/female)	80/82	71/79	83/77	64/74
Body weight of live pups (g) (mean +/- SD) on day 0 Males Females	6.2 +/- 0.5 9.4 +/- 1.2	6.1 +/- 0.4 9.5 +/- 1.1	6.0 +/- 0.4 9.1 +/- 0.7	6.3 +/- 0.5 9.9 +/- 0.9
Body weight of live pups (g) (mean +/- SD) on day 4 Males Females	6.0 +/- 0.4 9.0 +/- 1.3	5.9 +/- 0.4 9.2 +/- 1.1	5.8 +/- 0.3 8.8 +/- 0.5	6.0 +/- 0.5 9.5 +/- 1.0
Viability index	98.15	94.00	81.88	93.48
No. of external anomalies	0	0	0	0

 

Gestation index = (Number of dams with live newborns/Number of pregnant females) x 100

Birth index = (Number of newborns/Number of implantations) x 100

Viability index = (Number of live newborns on day 4 after birth/Number of live newborns) x 100

 

*: P<0.05 significantly different from control

 

Background level of stillborn : 0-14.84%

Background level of birth index : 80.98-96.61%

 

For further information, see "Attachments".

Conclusions:

The NOAEL is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring.

Executive summary:

In a combined repeated dose toxicity/reproduction and developmental toxicity study tetrahydromethylphthalic anhydride (MTHPA, 99.7%) was administered to groups of Crj:CD(SD) rats (12/sex), via gavage in corn oil at dose levels of 0 (Vehicle), 30, 100 and 300 mg/kg bw/day.

As for reproductive performance, no effects related to the test article were observed on the estrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices. Examination at delivery and during the lactation period revealed, no effects related to the test article in terms of gestational days, litter size and live newborns, gestation index, stillborn index, birth index, sex ratio, body weights of offspring at birth and at day 4 after birth, or viability index on day 4. No external anomalies were apparent. The NOAEL is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring.

This study is acceptable and satisfies the guideline requirement for a combined repeated dose toxicity/reproduction and developmental toxicity study in rats (OECD 422).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Well described GLP compliant study conducted to recognized international test guidelines.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study:

 

OECD 422

In a combined repeated dose toxicity/reproduction and developmental toxicity study tetrahydromethylphthalic anhydride (MTHPA, 99.7%) was administered to groups of Crj:CD(SD) rats (12/sex), via gavage in corn oil at dose levels of 0 (Vehicle), 30, 100 and 300 mg/kg bw/day.

As for reproductive performance, no effects related to the test article were observed on the estrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices. Examination at delivery and during the lactation period revealed, no effects related to the test article in terms of gestational days, litter size and live newborns, gestation index, stillborn index, birth index, sex ratio, body weights of offspring at birth and at day 4 after birth, or viability index on day 4. No external anomalies were apparent. The NOAEL is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring.

This study is acceptable and satisfies the guideline requirement for a combined repeated dose toxicity/reproduction and developmental toxicity study in rats (OECD 422).

 

 

 

Supporting study:

 

OECD 421

Furthermore, the effects of the structural analogue hexahydro-4-methylphthalic anhydride (4-MHHPA) on fertility, pregnancy and early lactation of the offspring have been investigated in a reproduction/developmental toxicity screening study conducted according to OECD 421. Groups of rats were dosed by oral gavage at levels of 50, 150 and 450 mg/kg/day. Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods and for 4 days post partum. No significant changes and no treatment-related effects were detected in males and females during the in vivo phase. Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls. Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment. Findings at macroscopic and microscopic examinations, in both males and females, did not reveal differences between groups that could be related to treatment. On the basis of these findings, 450 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL).

 

 

Waiving

An extended one generation reproduction toxicity study (e.g. according to OECD 443) was waived for the following reasons:

The available data for structural homologues of MTHPA indicate neither potential for teratogenic effects nor for reproduction toxicity in different species. These data together with the available information of the OECD 407, 408 and OECD 422 study allow a scientific validated evaluation of the respective endpoints and further tests would not be in line with animal welfare ideas.

 

 

Conclusion:

As an overall conclusion on the basis of the absence of adverse effects in the study for the test item as well as the structural analogue 4-MHHPA, a NOAEL of at least 300 mg/kg/day for reproductive performance of parents and for development of offspring was determined.

 

Effects on developmental toxicity

Description of key information

Based on a prenatal developmental toxicity study according to OECD 414 with the read-across substance 4 -MHHPA in rats, the NOAEL is considered to be 615 mg/kg/day for overall developmental toxicity including teratogenicity.

 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-06 to 2021-05-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: females: ca. 9 weeks of age at start of the mating period; males: 28-29 weeks of age at start of the mating period
- Weight at study initiation: females: 177-249 g
- Fasting period before study: no
- Housing: before mating: 1-2 females/cage, 2 males/ cage; during mating: 1 male and 1-2 females/cage; during gestation: 2 sperm positive females/ cage, if not possible 1 sperm positive female/ cage; cages: Type II polypropylene/polycarbonate; bedding: Safe 3/4-S Fasern Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany)
- Diet: ad libitum, ssniff SM R/M " breeding and maintenance autoclavable complete feed for rats and mice " produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: females: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 24.5
- Humidity (%): 37 - 62
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

Sunflower oil (Helianthi annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 115 mg/mL, 153.75 mg/mL and 200 mg/mL in the laboratory of Toxi-Coop Zrt daily to every three days.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil was used as vehicle, because the substance is hydrolytically unstable in aqueous media. Sunflower oil proved to be suitable for treatment.
- Concentration in vehicle: 115, 153.75, 200 mg/mL
- Amount of vehicle: A constant treatment volume of 4 mL dose preparation/kg body weight was administered in all groups.
- Lot/batch no.: 8006332001

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of the Test Facility two times during the study by a GC-MS method. Five samples from each concentration were taken from different places of the test item formulations for analysis of concentration and homogeneity. Similarly, five samples were taken from the vehicle (Control) and analyzed.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male/ one to two females
- Length of cohabitation: 2-4 h
- After unsuccessful pairing replacement of first male by another male with proven fertility: not performed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

14 days
The sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

daily

Duration of test:

The animals were sacrificed by anaesthesia on gestation day 20.

Dose / conc.:

800 mg/kg bw/day (nominal)

Dose / conc.:

615 mg/kg bw/day (nominal)

Dose / conc.:

460 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

A total of 104 males and 130 females for mating were used to achieve the sufficient number of females per group of at least 22 spermium positive females per group or at least 16 litters per group (28 to 30 spermium positive females and 19-23 litters per group achieved).

The number of mated and pregnant females was as follows:
Control: mated: 28, pregnant: 23
460 mg/kg bw/d: mated: 28, pregnant: 21
615 mg/kg bw/d: mated: 30, pregnant: 20
800 mg/kg bw/d: mated: 28, pregnant: 23

NB: Total pre-implantation losses were accounted as non-pregnant.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finding study, where oral treatment of pregnant Han: WIST rats with the dose level of 900 mg/kg bw/day of the test item caused death of three dams and clinical signs of the animals, as well as moderate reduction of food consumption and lower body weight gain. The increase of fetal death in the 900 mg/kg bw/day dose group due to one female with total post-implantation loss and moderately lower mean number of viable fetuses was considered treatment related, most likely secondary to the severe maternal toxicity. Less significantly lower food consumption and body weight gain including corrected body weight gain at 700 mg/kg bw/day were not unambiguously attributed to an effect of the test item. Slightly lower body weight of fetuses at 900 and 700 mg/kg bw/day (latter only females) was not proved to be due to the treatment with the test item. The test item caused neither malformations, nor increase of fetal variations or placental abnormalities.
A dose level of 800 mg/kg bw/day was selected as highest dose level in this main study with the aim to induce some maternal/developmental toxicity but not death or severe suffering. The low dose of 460 mg/kg bw/day was chosen to induce no toxic effect. The mid dose of 615 mg/kg bw/day was interpolated geometrically.
- Rationale for animal assignment (random): The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. If possible, females paired with the same male were allocated to different groups on the same mating day.
- Fasting period before blood sampling for dam thyroid hormones: No fasting period described.
- Time of day for dam blood sampling: in the morning on the day of necropsy

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day from g.d. 0 to 20, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were observed more frequently.
Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.
Observations for signs of morbidity and mortality were made twice daily from g.d. 5 to 19, at the beginning and end of the working period, and once on g.d. 20.

DETAILED CLINICAL OBSERVATIONS: Yes, see above.

BODY WEIGHT: Yes for females
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: 20
- Organs examined: The uterus with cervix and the left ovary removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed.
Thyroid glands, together with the parathyroid glands were removed and fixed in 4 % (v/v) buffered formalin solution for histopathological evaluation. They were weighed and recorded with a precision of 0.001 g after fixation.
Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % (v/v) buffered formalin solution at necropsy for possible histological examination. Control organs were fixed in 4 % (v/v) buffered formalin solution and preserved for comparison.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Live fetuses and fetal death were counted.

Blood sampling:

Blood samples collected for TSH and Thyroid Hormones (FT3 and FT4) measurements were drawn from all sperm positive females from the sublingual vein in the morning on the day of necropsy.
- Plasma: No
- Serum: Yes
- Volume collected: at least 1.0 mL blood

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter
- Anogenital distance of each fetus was determined.

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
Means and standard deviations and/or percentages were calculated.
The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Indices:

- Pre-implantation loss: ((Number of corpora lutea - Number of implantations)/Number of corpora lutea)*100
- Post-implantation loss: ((Number of implantations - Number of live fetuses)/Number of implantations)*100
- Sex distribution: (Number of Male (Female) fetuses/Number of fetuses)*100
- External abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses)*100
- Visceral abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses examined)*100
- Skeletal abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses examined)*100

Historical control data:

Historical control data on prenatal developmental toxicity from studies in the rat strain conducted in the same laboratory from 2017-2021 were included in the study report and used as reference.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Digging in the bedding after treatment, salivation, hunched back, piloerection, reduced activity and noisy breath were observed in the 615 and 800 mg/kg bw/day groups with moderate incidences. Hunched back and noisy breathing were observed also in one animal in the 460 mg/kg bw/day group. Dyspnoea was recorded for one female in the 615 mg/kg bw/day group. The clinical signs described for the 615 and 800 mg/kg bw/day groups were considered adverse and attributed to the treatment with the test item. The single finding in one animal of the low dose group is considered incidental.
In addition, one control female had swelling in the armpit without a toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female died in the course of the study in the 800 mg/kg bw/day group. It was found dead in the morning on g.d. 12. This female had clinical signs such as hunched back and piloerection from g.d. 10 onwards as well as reduced activity on g.d. 11. Digging in the bedding was also observed from g.d. 9 to 11.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was statistically significantly (p<0.01) lower in the 800 mg/kg bw/day group between g.d. 8 and 11, as well as g.d. 5 and 20 (p<0.05). Also, the corrected body weight gain was statistically significantly lower in this group (p<0.05). Statistics revealed also significance (p<0.05) in the 615 mg/kg bw/day group between g.d. 5 and 20. The statistically more pronounced lower body weight gain in the 800 mg/kg bw/day group between g.d. 8 and 11 was largely related to the female found dead on g.d.12. Overall, the lower body weight gain in the 800 and 615 mg/kg bw/day groups was attributed to an effect of the test item.
No differences in the 460 mg/kg bw/ day group were detected. Furthermore, no significant differences either in the body weight (including corrected body weight) of the dams of all treatment groups were observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower food consumption was observed in the 800, 615 and 460 mg/kg bw/day group in a dose-dependent manner between g. d. 8 and 11 (p<0.01 (-10 and -22 %) in the 460 and 800 mg/kg bw/day groups respectively and p<0.05 (-14 %) in the 615 mg/kg bw/day group). The reduction in the 800 mg/kg bw/day group between g.d. 8 and 11 was mostly caused by the female found dead on g.d.12. Between g.d. 11 and 14 statistical significance was indicated only in the 460 (-8 %) and 615 (-12 %) mg/kg bw/day groups and not in the 800 mg/kg bw/day group. Between g.d. 14 and 17 the values were statistically significantly lower only the 615 mg/kg bw/day group (p<0.05, -8 %) without a dose response.
The reduction in the 460 mg/kg bw/day dose group could be largely related to the low individual data of female No.: 181 between g.d. 5 and 11 which also exhibited clinical signs as hunched back on g.d. 10.
No effects on food consumption were observed at the other gestation days or groups.
If the percentages were taken into account, the reduced food consumption in the 800 mg/kg bw/day group between g.d. 8 and 11 was considered as a significant test-item related effect (-22%).
The lower food consumptions between g.d. 8 and 14 in the 615 mg/kg bw/day group and between g.d. 11 and 14 in the 800 mg/kg bw/day group were judged as slight or moderate effects of the test item (between 11 and 14 %).
The slight differences in the 460 mg/kg bw/day group between g.d. 8 and 14 were considered as not adverse, considering that the percentages were between 8 and 10 %.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower values were measured in the 800 (p<0.05, -13 %), 615 (p<0.01, -15 %) and 460 (p<0.01, -16 %) mg/kg bw/day groups for the free thyroid hormone level FT4. No dose-dependence was observed and all values were within the range of the historical control data (mean of 0.0129 and 22%CV). Therefore, these differences were considered to be indicative of biological variation and have no toxicological relevance. The FT3 values were also statistically significantly lower in the 615 and 460 mg/kg bw/day groups (p<0.01 both), but not in the 800 mg/kg bw/day group, hence without a dose response. The TSH hormone levels were similar in all groups. As no dose response relationship was observed for FT3 and TSH levels, the described effects are considered to be not test item related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean thyroid weight values were similar in the experimental groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lung emphysema, congestive liver and bloody jelly content in the ileum was recorded for one animal treated with 800 mg/kg bw/day which died before scheduled necropsy on gestation day 12.
Other findings such as pinhead-sized haemorrhages in the lungs, dilated renal pelvis occurred with low incidences and without a dose response. Therefore, they were considered as unrelated to the test item. In the control group a liver that was fused with the diaphragma or a subcutan formation was observed in one dam each.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item- related lesions observed upon histological examinations of the thyroid tissue.
The histological examination of the animal which died before scheduled necropsy in the 800 mg/kg bw/day group revealed multifocal myocardial fibroblast proliferation accompanied with atrophy of affected myocytes in the heart, alveolar emphysema in the lungs, and congestion in the liver as well as in the small intestines as cause of death. Lung emphysema, congestive liver and bloody jelly content in the ileum was recorded for one animal treated with 800 mg/kg bw/day which died before scheduled necropsy on gestation day 12. Findings such as pinhead-sized haemorrhages in the lungs, dilated renal pelvis occurred with low incidences and without a dose response. Therefore, they were considered as unrelated to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

One female in the 800 and one in the 615 mg/kg bw/day group had total postimplantation loss. In the 615 mg/kg bw/day group two females and in the 800 mg/kg bw/day group as well as in the control one female had total preimplantation loss.
If the females with total pre- and post-implantation loss were included to the data evaluation, the post-implantation loss was statistically significantly higher in the 800 mg/kg bw/day group (for mean value: p<0.05 by U-test and for sum p<0.01 by Chi square).
According to the Chi square test also early embryonic death (sum) increased statistically significantly (p<0.01) for those animals. Preimplantation loss and intrauterine mortality (summarized pre- and post-implantation loss) were statistically significantly higher (p<0.01) for sum value in the 615 mg/kg bw/day group. No dose dependency was observed.
Statistically significantly lower sum values were seen in the 460 mg/kg bw/day group (pre-implantation loss and total intrauterine mortality; p<0.05 by Chi square) without a toxicological relevance. There were no statistically significant differences in the mean number of implantations and late embryonic death.
If the females with total pre- and post-implantation loss were excluded, difference from the control values decreased and there was no statistical significance indicated in any parameters.
For the slight increase of post-implantation loss (early and late resorptions) including the occurrence of total post-implantation loss in one female both in the 800 and 615 mg/kg bw/day group a treatment relationship cannot be excluded.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "Pre- and post-implantation loss".

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "Pre- and post-implantation loss".

Dead fetuses:

no effects observed

Description (incidence and severity):

No fetal death was observed in all groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number and percentage of pregnant females compared to mated females showed similar values in all groups.
Control: mated: 28, pregnant: 23/ 82.1%
460 mg/kg bw/d: mated: 28, pregnant: 21/ 75.0 %
615 mg/kg bw/d: mated: 30, pregnant: 20/ 66.7 %
800 mg/kg bw/d: mated: 28, pregnant: 23/ 82.1 %

Other effects:

no effects observed

Description (incidence and severity):

The values for placental weight were similar in the treatment groups and the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the body weight of the fetuses. The values for body weight were similar in the treatment groups and the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses was equal among the groups.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The values for absolute and normalized anogenital distance were similar in the treatment groups and the control group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of evaluated fetuses was 266, 243, 209 and 229 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively.

Variations
There was no variation observed.

Malformations
There were no malformations found.

Growth retardation
The incidence of body weight retarded fetuses was higher in the 800 and 615 mg/kg bw/day groups (at a similar level), however statistics revealed no significance. Hence, a test item effect was not proved.

Placentas
Larger placentas and or with wider margin were found in one litter in the 800 mg/kg bw/day group. A test item relationship remains unclear as this observation was limited to only one single dam. It should be noted, that two fetuses to which these placentas belonged were malformed (bent scapula or and slightly thicker humerus).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 129, 122, 104 and 114 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. The incidence of fetuses with abnormalities and variations was statistically significantly higher (p<0.05 and p<0.01 respectively) in the 615 mg/kg bw/day group according to the Chi square test. No statistical difference was observed if the number of litters affected was considered. The mean percent per litter for total variations was also significantly higher (p<0.05) according to Duncan's multiple range test in the 615 mg/kg bw/day group. There was no dose response indicated in variations, abnormalities and in the incidence of malformations. The incidence of malformations was lower than in the control group at 460 mg/kg bw/day (p<0.01 according to the Chi square test) and at 615 mg/kg bw/day. The number of fetus with malformations was comparable to control in the
800 mg/kg bw/day group.

Variations, malformations:
Statistically significantly higher incidence (p<0.05) according to the Chi square test was seen in the 615 mg/kg bw/day group only for the number of affected fetuses with 1 or more bones incompletely ossified. If only fetuses with 3 or more incompletely ossified bones were evaluated no statistically significant differences were observed. This variation was at a similar level in the 800 mg/kg bw/day group however statistics revealed no significance.
Also, in case of less ossified metacarpal/metatarsal, the number of affected fetuses and litters was statistically significantly higher (p<0.01 and p<0.05 respectively) according to the Chi square test in the 615 mg/kg bw/day group but not in the 800 mg/kg bw/day dose group. The incidences were not statistically significant when the litter means were compared. There were slightly higher incidences in case of some variations (retarded skull bones and unossified hyoid at 800 mg/kg bw/day, ossified sternebra and incomplete ossification of vertebra (SII or from SII) at 615 and 800 mg/kg bw/day), however without a statistical significance.
These increases were judged to be borderline cases considering the lack of clear statistical significances and the slight differences/low incidences. The slight differences might be related to reduced body weight gain and food consumption between g.d. 8 and 14 in the 800 and 615 mg/kg bw/day groups.
Slightly bent scapula (variation) occurred only in the test item treated groups (with an incidence of 1, 3 and 1 in the 460, 615 and 800 mg/kg bw/day groups, respectively) without a dose response. Two more fetuses (in one litter) had bent scapula (one with slightly thicker humerus) in the 800 mg/kg bw/day group, which was considered as a malformation. It should be noted, that those two fetuses belonged to the changed placentas, as detailed under external examinations.
The incidence of slightly bent or bent scapula as well as malformed humerus was low and may occur in control fetuses according to the historical control data, hence a test item response was not proved.

There was no indication on a test item effect in the 460 mg/kg bw/day group.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 137, 121, 105 and 115 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. There were no statistically significant increases in the incidence of visceral variations and malformations.

Variations:
Slightly dilated ventricles were recorded in the control and 615 mg/kg bw/day group in a few fetuses. However, the number of fetuses with the dilated ventricles was highest in the control group and no dose- response relationship was observed. Slightly enlarged peri-meningeal space was observed in one fetus in the 460 mg/kg bw/day group. Bilateral hydroureter were found in all groups without a dose-response. Hydroureter accompanied with dilated renal pelvis was observed in all groups without a statistically significant increase. A slightly malpositioned kidney was found in one control fetus.
Considering the lack of dose- response and the low incidence the above variations were not attributed to the treatment.

Malformations
Brain malformations were found in one fetus each of the mid and high dose group. One fetus in the 800 mg/kg bw/day group had external hydrocephaly and a hole in the thalamus region was found in cased of one fetus in the 615 mg/kg bw/day group.
Taking into account that external hydrocephaly and partial deficiency of brain tissue may occur in control fetuses with low incidences according to the background data of this laboratory, and the low incidences (one each at 615 and at 800 mg/kg bw/day group), the occurrence of these malformations was considered unrelated to the treatment with the test item.

Other effects:

no effects observed

Description (incidence and severity):

Fetal Examination (External, Visceral, Skeletal) - Overall
The number of evaluated litters/fetuses was 23/266, 21/243, 19/209 and 20/229 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively.
There were 4/23, 0/21, 2/19 and 5/20 litters with malformed fetuses respectively. There were no statistically significant differences in the incidence of malformations over all dosing groups.

Key result

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related adverse effect on the fetuses were observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

615 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as to be 460 mg/kg bw/day for maternal toxicity and and 615 mg/kg bw/day for overall developmental toxicity including teratogenicity.

Executive summary:

The test item was examined for its possible prenatal developmental toxicity in a study according to OECD 414. Groups of 28 to 30 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 460, 615 and 800 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 28 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 280 mg/g. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 91 and 105 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 23, 21, 19 and 20 evaluated litters in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. One female died in the course of the study in the 800 mg/kg bw/day group due to an effect of the test item after clinical signs such as hunched back, piloerection and reduced activity and digging in the bedding in the days before. Necropsy revealed macroscopic changes in the lungs, liver and ileum. Histopathology supported these findings and additionally there were lesions in the heart observed. Similar test item related clinical signs were observed in the 800 and 615 mg/kg bw/day group. Reduced food consumption and lower body weight gain (including corrected body weight gain in the 800 mg/kg bw/day dose group) was observed in the 800 and 615 mg/kg bw/day group which was attributed to an effect of the test item. Slight reductions in the food consumption of the 460 mg/kg bw/day group animals were not considered as adverse. There were no significant differences in the body weight (including corrected body weight) of the dams. Lower values compared to control were measured in all dose groups (in similar manner) for the free thyroid hormone level FT4. The FT3 values were also statistically significantly lower in the 615 and 460 mg/kg bw/day groups. In the absence of a dose-response relationship these findings are considered not test-item related. The TSH hormone levels were similar in all groups. There were no test item related differences in thyroid weight among the dosing groups. The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Slight increase of post-implantation loss (early and late resorptions) including the occurrence of total post-implantation loss in one female both in the 800 and 615 mg/kg bw/day group were likely attributed to an effect of the test item, possibly due to maternal toxicity. The fetal weight was slightly lower and the incidence of growth retarded fetuses was higher in the 800 and 615 mg/kg bw/day groups (at a similar level), however statistical analysis revealed no significance. Hence, a test item effect was not proved. There were no significant differences in the ano-genital distance and placental weight parameters. At fetal examinations, there were no significant differences in the incidence of malformations at any dose level. There were no malformed fetuses found on external examination. In one litter, the placentas were observed as larger and or with wider margin in the 800 mg/kg bw/day group. Considering the lack of dose response or the low incidences, the visceral variations observed were not attributed to treatment. Two single cases of brain malformations such as an external hydrocephaly and a hole in the thalamus region in the dose groups of 800 and one 615 mg/kg bw/day group were considered incidental and toxicologically not relevant. Some increases in delayed ossifications might be related to maternal toxicity in the 800 and 615 mg/kg bw/day groups. Slightly bent scapula occurred in the test item treated groups with low incidences (1 to 3 fetuses per dose group) and two fetuses of one litter had bent scapula (one with slightly thicker humerus) in the 800 mg/kg bw/day group (latter in the litter with placenta alterations). Slightly bent or bent scapula as well as malformed humerus may occur in control fetuses according to the background data, hence a test item response was not proved.

In conclusion the oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose level of 800 mg/kg bw/day caused test-item related death of one animal. Clinical signs of animals in the 800 and 615 mg/kg bw/day groups were attributed to the treatment. The treatment with the test item caused reduced food consumption and body weight gain in the 615 and 800 mg/kg bw/day groups. Besides a significantly decreased corrected body weight gain in the 800 mg/kg bw/day group no significant differences in the body weight/corrected body weight values were detected. Thyroid-related parameters (FT3, FT4 TSH, weight, histopathology) were not affected by treatment. The slightly but statistically significantly increased postimplantation loss in the 615 and 800 mg/kg bw/day group is considered to be treatment-related. Although the incidence of body weight retarded fetuses was slightly increased in the mid and high dose group, there were no statistically significant differences in the mean body weight of the fetuses. No statistically significant differences in the incidence of malformations over all dosing groups were observed. Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as to be 460 mg/kg bw/day for maternal toxicity and and 615 mg/kg bw/day for overall developmental toxicity including teratogenicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

615 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Well described GLP compliant study conducted to recognized international test guidelines.

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 data were available on effects on the developmental toxicity of MTHPA, instead, a read across approach was applied using data from the structural analogue 4-MHHPA as similar chemical and toxicological properties are assumed. The experimental design and the results of the source-studies from the structural analogue 4-MHHPA are considered to cover the key parameters adequately, with an exposure duration comparable to or longer than the corresponding study. Therefore this data is deemed to be relibale and also adequate for the purpose of classification and labelling and/or risk assessment.

 

Key study:

 

OECD 414 (2021)

The test item was examined for its possible prenatal developmental toxicity in a study according to OECD 414. Groups of 28 to 30 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 460, 615 and 800 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 28 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 280 mg/g. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 91 and 105 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 23, 21, 19 and 20 evaluated litters in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. One female died in the course of the study in the 800 mg/kg bw/day group due to an effect of the test item after clinical signs such as hunched back, piloerection and reduced activity and digging in the bedding in the days before. Necropsy revealed macroscopic changes in the lungs, liver and ileum. Histopathology supported these findings and additionally there were lesions in the heart observed. Similar test item related clinical signs were observed in the 800 and 615 mg/kg bw/day group. Reduced food consumption and lower body weight gain (including corrected body weight gain in the 800 mg/kg bw/day dose group) was observed in the 800 and 615 mg/kg bw/day group which was attributed to an effect of the test item. Slight reductions in the food consumption of the 460 mg/kg bw/day group animals were not considered as adverse. There were no significant differences in the body weight (including corrected body weight) of the dams. Lower values compared to control were measured in all dose groups (in similar manner) for the free thyroid hormone level FT4. The FT3 values were also statistically significantly lower in the 615 and 460 mg/kg bw/day groups. In the absence of a dose-response relationship these findings are considered not test-item related. The TSH hormone levels were similar in all groups. There were no test item related differences in thyroid weight among the dosing groups. The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Slight increase of post-implantation loss (early and late resorptions) including the occurrence of total post-implantation loss in one female both in the 800 and 615 mg/kg bw/day group were likely attributed to an effect of the test item, possibly due to maternal toxicity. The fetal weight was slightly lower and the incidence of growth retarded fetuses was higher in the 800 and 615 mg/kg bw/day groups (at a similar level), however statistical analysis revealed no significance. Hence, a test item effect was not proved. There were no significant differences in the ano-genital distance and placental weight parameters. At fetal examinations, there were no significant differences in the incidence of malformations at any dose level. There were no malformed fetuses found on external examination. In one litter, the placentas were observed as larger and or with wider margin in the 800 mg/kg bw/day group. Considering the lack of dose response or the low incidences, the visceral variations observed were not attributed to treatment. Two single cases of brain malformations such as an external hydrocephaly and a hole in the thalamus region in the dose groups of 800 and one 615 mg/kg bw/day group were considered incidental and toxicologically not relevant. Some increases in delayed ossifications might be related to maternal toxicity in the 800 and 615 mg/kg bw/day groups. Slightly bent scapula occurred in the test item treated groups with low incidences (1 to 3 fetuses per dose group) and two fetuses of one litter had bent scapula (one with slightly thicker humerus) in the 800 mg/kg bw/day group (latter in the litter with placenta alterations). Slightly bent or bent scapula as well as malformed humerus may occur in control fetuses according to the background data, hence a test item response was not proved.

In conclusion the oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose level of 800 mg/kg bw/day caused test-item related death of one animal. Clinical signs of animals in the 800 and 615 mg/kg bw/day groups were attributed to the treatment. The treatment with the test item caused reduced food consumption and body weight gain in the 615 and 800 mg/kg bw/day groups. Besides a significantly decreased corrected body weight gain in the 800 mg/kg bw/day group no significant differences in the body weight/corrected body weight values were detected. Thyroid-related parameters (FT3, FT4 TSH, weight, histopathology) were not affected by treatment. The slightly but statistically significantly increased postimplantation loss in the 615 and 800 mg/kg bw/day group is considered to be treatment-related. Although the incidence of body weight retarded fetuses was slightly increased in the mid and high dose group, there were no statistically significant differences in the mean body weight of the fetuses. No statistically significant differences in the incidence of malformations over all dosing groups were observed. Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as to be 460 mg/kg bw/day for maternal toxicity and and 615 mg/kg bw/day for overall developmental toxicity including teratogenicity.

 

 

Supporting studies:

 

DRF (2021)

The structural analogue hexahydro-4-methylphthalic anhydride (4-MHHPA) was examined for its possible prenatal developmental toxicity in a dose range finding study. Groups of eleven to twelve spermium-positive female Han: WIST rats were treated with the test item by oral administration daily at three dose levels of 500, 700 and 900 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of eleven spermium positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when spermium was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day (g.d.) 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally.

On gestation day 20, a total of 10, 9, 11 and 6 litters in the control, 500, 700 and 900 mg/kg bw/day groups, respectively, were evaluated. The dose level of 900 mg/kg bw/day caused death of three dams. One dam died after five, one after six and one after fourteen administrations. Reduced activity and piloerection were recorded for the animals that died and in addition in two females that survived (one with total post-implantation loss and one non-pregnant) in the 900 mg/kg bw/day group. Laying down and dyspnoe was recorded for one female which died. Digging in the bedding after treatment and salivation was observed in all test item treated groups. All of the three animals which died before scheduled necropsy had dark red liver. Bloody or yellow or yellowish-brownish content in the intestines or yellow dilute or whitish content in the stomach was recorded for the animals that died and for the surviving female with total post-implantation loss in the 900 mg/kg bw/day group. Sporadically gas filled intestine segments were observed in the animals in the 900 and 500 mg/kg bw/day group. In one high dose animal which died reddish mottled lungs were found. Slight to moderately lower mean food consumption of the dams was indicated, most notable between g.d. 5 and 11, in the 900 mg/kg bw/day group. In the 700 mg/kg bw/day dose group the average food consumption was slightly lower between g.d. 5 and 11. This was not unambiguously attributed to the test item. Slightly lower body weight was observed in the 900 and 700 mg/kg bw/day group, in the latter not proved to be due to the test item. Differences were more notable in regard to lower body weight gain values in the 900 mg/kg bw/day dose group and also observed in the 700 mg/kg bw/day dose group. In the 700 mg/kg bw/day dose group this effect was less pronounced. The slightly lower body weight gain in the 500 mg/kg bw/day between g.d. 5 and 8 was not proved to be due to the test item. The corrected body weight gain was lower in the 700 and 900 mg/kg bw/day group with a dose dependent response. There was no dose response indicated in the mean percent of early embryonic death. The percentage of late embryonic death increased slightly and the average fetal death increased due to one female with total post-implantation loss in the 900 mg/kg bw/day group. There was no test item effect indicated in the number of implantations or percent pre-implantation loss. The number of viable fetuses was slightly to moderately lower in the 700 and 900 mg/kg bw/day group, not proved to be due to the test item at 700 mg/kg bw/day. Slightly lower fetal weight in the 900 mg/kg bw/day group and in case of female fetuses in the 700 mg/kg bw/day group was not considered as significant. There were no malformations or placental alterations found. There was no test item effect indicated in incidence of variations i.e. growth retardations.

In the present dose range finding study, oral treatment of pregnant Han: WIST rats with the dose level of 900 mg/kg bw/day of the test item caused death of three dams and clinical signs such as reduced activity, piloerection, laying, dyspnoe and salivation of the animals. The test item caused moderate reduction in the food consumption and lower body weight parameters at 900 mg/kg bw/day (including a minimal weight loss between g.d. 5 and 8 and a moderately lower corrected body weight gain). Less significantly lower food consumption and body weight parameters including corrected body weight gain at 700 mg/kg bw/day were not unambiguously attributed to an effect of the test item. The increase of fetal death in the 900 mg/kg bw/day dose group due to one female with total post-implantation loss and moderately lower mean number of viable fetuses was considered treatment related, most likely secondary to the severe maternal toxicity demonstrated by the strong clinical symptoms. The mean number of implantations, percentage preimplantation loss, early and late resorptions were not proved to be affected by the test item. Slightly lower body weight of fetuses was not proved to be due to the treatment with the test item. The test item caused neither malformations, nor increase of fetal variations or placental abnormalities. Based on the observations the dose range finding study determined the doses of 460, 615 and 800 mg/kg bw/d for the main study (OECD 414 (2021)). The No Observed Adverse Effect Level (NOAEL) was considered to be 500 mg/kg bw/d for maternal toxicity and developmental toxicity.

 

Conclusion:

As an overall conclusion adverse effects caused by the test item in the dams offspring, this possibly due to the maternal toxicity, have been identified and the NOAEL for maternal and developmental toxicity including teratogenicity was determined to be 460 mg/kg bw/day for the structural analogue hexahydro-4-methylphthalic anhydride (4-MHHPA). According to the results of the read-across substance the NOAEL of 615 mg/kg bw/d is considered for the registration substance MHHPA for maternal and developmental toxicity.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available studies are considered reliable for this assessment.

 

Reproductive toxicity:

Based on the above stated assessment of the reproduction toxicity potential of the test item, it is deemed that the compound is not toxic to reproduction and does not require classification according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU and as amended for the eighteenth time in Regulation (EU) 2022/692.