Tetrahydromethylphthalic anhydride

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting 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 same 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

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/cage, 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

Vehicle:

corn oil

Details on oral 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 no.: V5P5831, nakalai tesque Co.

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

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

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: male: two times/week
female: two times/ week at pre-mating period, in pregant 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 "Attachments" field) were examined.: WBC, RBC, Hb, Ht, PLT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:after dosing period (same time for HEMATOLOGY)
- Animals fasted: Yes
- How many animals: All of male
- Parameters checked in table (see below in "Attachments" field) 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below in "Attachments" field)
HISTOPATHOLOGY: Yes (see below in "Attachments" field)

Other examinations:

no details given

Statistics:

no details given

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:

not specified

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):

Blood chemistry in males showed a decrease in total cholesterol and urea nitrogen and an increase in triglycerides in the 300 mg/kg group. The changes were considered as non-adverse, since the were of minor degree, a clear dose-repsonse was missing and no further collaborative findings were observed, e.g. no pathological changes in the kidneys or liver.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the male adrenal glands absolute weights increased in the 100 mg/kg bw/d group and relative weights in the 300 mg/kg bw/d group. The changes in the 100 mg/kg bw/d group were considered incidental as they were not observed in females nor in the high dose group. The changes in relative weight in the 300 mg/kg bw/d group were considered to be of no toxicological significance, as they were not observed in females and no abnormalities were found on histopathological examination.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pathological examination showed gross thickening of the mucosa of the forestomach in males and females in the 300 mg/kg bw/d group. These changes were considered to be inflammatory changes and reactive proliferation of the epithelium caused by the local irritant properties of the test substance. Grossly, no gastric changes were observed in the 100 mg/kg bw/d group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/d histological examination showed effect in the forestomach: hyperplasia and vacuolisation of the squamous epithelium, granulomatous inflammation of the squamous epithelium and submucosal tissue as well as erosions. These changes were considered to be inflammatory changes and reactive proliferation of the epithelium caused by the local irritant properties of the test substance. In the 100 mg/kg bw/d group, hyperplasia of the squamous epithelium of the forestomach was observed in one male.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Tabulated data are presented in the attached documents.

CLINICAL SIGNS AND MORTALITY
Three animals (male and female of 300 mg/kg group and male of 30 mg/kg group) is died by the accident at administration. Salivation was observed in 4-9(/12) animals at male 300 mg/kg on and after day 36. Salivation was observed immediately after administration, and continued about 30 min.

BODY WEIGHT AND WEIGHT GAIN
No stat. sig. difference from controls.

FOOD CONSUMPTION AND COMPOUND INTAKE
No stat. sig. difference from controls except one case (male 30 mg/kg, day 49, increase in consumption).

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
No stat. sig. difference from controls.

CLINICAL CHEMISTRY
Males: Decrease of total cholesterol and BUN, increase of triglyceride at 300 mg/kg (p<0.05). Decrease of A/G ratio at 100 mg/kg (p<0.05). All changes were considered to be either incidental or of no toxicological relevance (i.e. non-adverse), since the were of minor degree, a clear dose-repsonse was missing and no further collaborative findings were observed.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Male: Increase in kidney weight and adrenal weight at 100 mg/kg (absolute) (p<0.05), Increase in adrenal weight at 300 mg/kg (relative) (p<0.05). The changes were considered to be either incidental or of no toxicological relevance (i.e. non-adverse) since there were limited to one sex and no collaborative findings were noted.
Female: No stat. sig. difference from controls

GROSS PATHOLOGY
hyperplasia (male:11/11, female 8/12) at forestomach mucosa (in 300 mg/kg, terminal sacrifice)

HISTOPATHOLOGY: NON-NEOPLASTIC
Limited to findings in the forestomach: At 300 mg/kg bw/d hyperplasia and vacuolisation of the squamous epithelium, granulomatous inflammation of the squamous epithelium and submucosal tissue as well as erosions. These changes were considered to be inflammatory changes and reactive proliferation of the epithelium caused by the local irritant properties of the test substance. In the 100 mg/kg bw/d group, hyperplasia of the squamous epithelium of the forestomach was observed in one male.

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:

Key result

Dose descriptor:

NOEL

Remarks:

local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: hypertrophy of Gastric mucosa, 1/12 at 100 mg/kg bw/d

Key result

Dose descriptor:

NOEL

Remarks:

local toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: hypertrophy of Gastric mucosa, 9/12 at 300 mg/kg bw/d

Key result

Critical effects observed:

no

The study authors only derived NOELs (males: 30 mg/kg bw/d, females: 100 mg/kg bw/d). Changes potentially indicative of systemic toxicity were only oberved at 300 mg/kg bw/d. However they were either incidental or of no toxicological relevance (i.e. non-adverse). Therefore a NOAEL (systemic toxicity) of 300 mg/kg bw/d can be derived for both sexes.

Due to the effects observed at necropsy and histopathology in the forstomach, the NOEL/NOAEL (local toxicity) is 30 mg/kg bw/d (males) and 100 mg/kg bw/d (females).

Conclusions:

A NOAEL of 300 mg/kg bw/d for systemic toxicity and a NOEL/NOAEL of 30 (males) and 100 (femlaes) mg/kg bw/d was determined in a combined repeated dose/reproduction toxicity screening study (OECD 422) in rats.

Executive summary:

Tetrahydromethylphthalic anhydride (MTHPA) was administered by gavage at doses of 0, 30, 100 and 300 mg/kg/d for 49 days in males and from 14 days before mating to day 3 of lactation in females (total number of 38 days). All animals survived at all treated group, except three animals died by accident (one female in 30 mg/kg bw/d, one male in 300 mg/kg and one female in 300 mg/kg group). Salivation was transiently observed in males of 300 mg/kg bw/d group at the day 36-49. Histopathological examination revealed squamous hyperplasia of the forestomach in both sexes of the 300 mg/kg bw/d group, epithelial vascular change, edema and cellular inflammation of the forestomach in males of the 300 mg/kg bw/d group, and erosion of the forestomach in females of 300 mg/kg bw/d group. There were no adverse effects on body weight and food consumption. There were no alterations related to tetrahydromethyl-1, 3-isobenzofuranedione on hematological examination. Slightly decreased total cholesterol and BUN and increased triglyceride were observed in males of the 300 mg/kg bw/d. They were considered as non-adverse since they were of minor degree and no collaborative findings occurred. As a gross finding, mucosal thickening of the forestomach was found in both sexes of the 300 mg/kg bw/d group. In the male adrenal glands absolute weights increased in the 100 mg/kg bw/d group and relative weights in the 300 mg/kg bw/d group. The changes in the 100 mg/kg bw/d group were considered incidental as they were not observed in females nor in the high dose group. The changes in relative weight in the 300 mg/kg bw/d group were considered to be non-adverse, as they were not observed in females and no abnormalities were found on histopathological examination.

The major toxicity was inflammation of stomach mucosa. On the basis of this study, the NOAEL of 300 mg/kg bw/d can be derived for systemic toxicity and the NOEL/NOAEL of 30 (males) and 100 (femlaes) mg/kg bw/d for local toxicity.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydromethylphthalic anhydride

EC Number:

234-290-7

EC Name:

Tetrahydromethylphthalic anhydride

Cas Number:

11070-44-3

Molecular formula:

C9H10O3

IUPAC Name:

3a-methyl-3a,4,5,6-tetrahydro-2-benzofuran-1,3-dione

Details on test material:

- Name of test material (as cited in study report): tetrahydromethyl-1,3-isobenzofurandione
- Lot/batch No.: 2522
- Stability under test conditions: stable, Vehicle: Corn oil (nakaraitesk, lot No, V5P5523)
- Storage condition of test material: room temperature, dark place

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 4

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

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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".

Applicant's summary and conclusion

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).