1,2,3,6-tetrahydro-3-methylphthalic anhydride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

key study

Study period:

1995.12.6-1997.6.5

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

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: 356.3-394.4g, female: 213.5-252.9g
- Fasting period before study: 18hr
- Housing: dosing period: stainless hanger gage, one animal/cage, mating period: polycarbonate gage with wood chip
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26°C
- 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 (if other than water): Corn oil is used generally
- Concentration in vehicle: 0.6, 2 and 6w/v%
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no. (if required): V5P5831, nakalai tesque Co.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The prepared test solution was analysed at the start week of administration, and it was confirmed that it was within the permissible range (± 5%) of the nominal concentration.

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 (nominal)

Remarks:

Vehicle control

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: oral, one of the identical exposure route for humans

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 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 (see below in remarks 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
HISTOPATHOLOGY: Yes

Other examinations:

no details given

Statistics:

Body weight, food consumption, haematology values, blood chemistry values, mating days, sexual cycle test value (number of estrus), organ weight, gestation period, number of corpora lutea, number of implantation marks, number of pups born - average value and standard deviation were calculated for each group. First, the homogeneity of dispersion among the substance administration groups was tested by the Bartlett method. When the variances were uniform, the Dunnett multiple comparison test was used to compare with the control group, and when the variances were not uniform, the Steei multiple comparison test was used to compare with the control group.
The copulation rate, fertility rate, birth rate and sex ratio of offspring are determined by the χ 2 test, and the stillbirth rate, birth rate and 4-day survival rate determined by the Wilcoxon rank sum test. The control group and each treatment group were compared. In all cases, significance level was set to 5%. The measured values for the offspring were processed in litter units.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males in the 300 mg/kg group, salivation was observed from 36 days after administration. Approximately 9 cases were observed. Recovery had occurred within 30 minutes after dose administration.
In addition, hair loss was noted in individual males and females in the control group and in 1 female in the 100 mg/kg group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 300 mg/kg group died immediately after administration on Day 4 immediately after dosing as did one female in the 30 mg/kg group on Day 13 after dosing. In the 300 mg/kg group, 1 female showed decreased activity, slow breathing, pale skin, swelling of the abdomen, and dirt in the lower abdomen after 17 days of administration. The animal was humanely killed. Necropsy/tissue histopathology was performed.
At necropsy of these deaths, dark redness of the lungs, constriction of the lungs, foamy fluid retention in the trachea, black-red spots of the oesophagus. Fat droplets, which were considered to be corn oil, were found in the alveoli, it was concluded that the cause of death was the error in administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no difference in body weight between male and female in each group compared with the control group throughout the administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Relative to controls, food intake was statistically significantly increased in males in the 30 mg/kg group on Day 49 of administration.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No difference was observed between each treatment group and the control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the 300 mg/kg group, a statistically significant decrease in total cholesterol and urea nitrogen and an increase in triglyceride were observed. A statistically significant decrease in the A/G ratio was observed in the 100 mg/kg group. These were all mild changes.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, a statistically significant increase in absolute adrenal weight was observed in the 100 mg/kg group and relative adrenal weight was statistically significantly increased in the 300 mg/kg group. In addition, a statistically significant increase in absolute kidney weight was observed in the 100 mg/kg group.
No change was observed in females in each group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy of males after the end of the treatment period, thickening of the forestomach was observed in all animals of the 300 mg/kg group. Hair loss was observed in one individual of the control group.
In females, thickening of the mucosa of the forestomach was observed in 8 animals of the 300 mg/kg group. In addition, hair loss was observed in 1 case each in the control group and 100 mg/kg group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, animals of the 300 mg/kg group showed hyperplasia of the squamous epithelium of the forestomach and granulomatous inflammation of the submucosa. Vacuolarization of the squamous epithelium and oedema of the squamous subepithelial tissue from the squamous epithelium were observed in 10 cases, and hyperplasia of the squamous epithelium of the forestomach was observed in 1 animal of the 100 mg/kg group. In addition, erosion of the integument was found on the skin in 1 male control group with hair loss.
In females, hyperplasia of the anterior gastric forestomach epithelium, granulomatous inflammation of the submucosa was seen in 7 animals and erosion in 2 animals of the 300 mg/kg group. In addition, atrophy of the thymus was observed in one animal of the 300 mg/kg group. However, similar changes were observed in one animal of the control group.
One of the 2 animals of the 100 mg/kg group autopsied because all of the offspring died had one lung. Cellular infiltration mainly of neutrophils and atrophy of the thymus were observed, but no abnormality was observed in the other case.
In the case of death of the male in the 300 mg/kg group due to administration error, the common changes were oedema, neutrophils, and histiocytes from the squamous epithelium of the forestomach to the submucosa. In addition to the presence of infiltrates, pulmonary haemorrhage and oedema were observed in the one deceased male, and haemorrhage of the oesophageal muscle layer and cellular infiltration mainly of neutrophils in the one female of this group following humane kill from the study. Atrophy of the thymus and necrosis of the tubular epithelium in one part of the renal cortex were observed. In addition, in one female in the 30 mg/kg group which died due to administration error, congestion of the lungs, edema, aggregation of foam cells, infiltration of neutrophils, lymphocytes, etc., and interstitial fluid in the bronchiole lumen and thymic atrophy was observed.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: hypertrophy of Gastric mucosa, 1/12

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: hypertrophy of Gastric mucosa, 9/12

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: hypertrophy of Gastric mucosa, 12/12

Critical effects observed:

not specified

NOAEL : Male = 100 mg/kg bw

Female = 100 mg/kg bw

Relative and/or absolute adrenal and/or kidney(s) weigh increase was observed in male 100 and/or 300mg/kg group, but no histopathological change was observed, so the author excluded this observation. Squamous hyperplasia was observed in only one male of 100 mg/kg group, and 8/12 females of 300mg/kg.

Therefore, the NOAEL was determined to be 100mg/kg in both sex.

Conclusions:

A NOAEL of 100 mg/kg bw/day was determined in a combined repeated dose/reproduction toxicity study (OECD 422) in rats based on decreased clinical chemistry parameters, changes in organ weights and inflammation of the forestomach mucosa.

Executive summary:

Tetrahydromethylphthalic anhydride (MTHPA) was administered by gavage at doses of 0, 30, 100 and 300 mg/kg/day 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, one male in 300 mg/kg and one female in 300 mg/kg group). Salivation was transiently observed in males of 300 mg/kg group at the day 36-49. Histopathological examination revealed squamous hyperplasia of the forestomach in both sexes of the 300 mg/kg group, epithelial vascular change, edema and cellular inflammation of the forestomach in males of the 300 mg/kg group, and erosion of the forestomach in females of 300 mg/kg 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. Decreased total cholesterol and BUN and increased triglyceride were observed in males of the 300 mg/kg. As a gross finding, mucosal thickening of the forestomach was found in both sexes of the 300 mg/kg group. Increased adrenal weights were observed in males of the 300 mg/kg group.

The major toxicity was inflammation of stomach mucosa. On the basis of this study, NOEL is 30mg/kg/day (male), 100mg/kg/day (female) and NOAEL is considered to be100 mg/kg/day for both sexes.

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

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
- Analytical purity: 99.7%
- 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
- Molecular formula (if other than submission substance): C9 H10 O3
- Molecular weight (if other than submission substance): 166.18
- Smiles notation (if other than submission substance): O=C(OC(=O)C1CC=C(C2)C)C12
- InChl (if other than submission substance): 1/C9H10O3/c1-5-2-3-6-7(4-5)9(11)12-8(6)10/h2,6-7H,3-4H2,1H3
- Structural formula attached as image file (if other than submission substance): see Fig. (11070-44-3 structure.jpg)

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: 356.3-394.4g, female: 213.5-252.9g
- Fasting period before study: 18hr
- Housing: dosing period: stainless hanger gage, one animal/gage, mating period: polycarbonate gage with wood chip
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26°C
- 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 (if other than water): Corn oil is used generally
- Concentration in vehicle: 0.6, 2 and 6w/v%
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no. (if required): 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:

yes

Details on analytical verification of doses or concentrations:

The prepared test solution was analysed at the start week of administration, and it was confirmed that it was within the permissible range (± 5%) of the nominal concentration.

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.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Doses of 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: oral, one of the identical exposure route for human
High dose of 300 mg/kg based on outcome of preliminary study. Lower doses of 100 and 30 mg/kg set at a common ratio 3 from the selected high dose level of 300 mg/kg

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 HEMATOLOGY)
- Animals fasted: Yes
- How many animals:All of male
- Parameters checked in table (see below in remarks 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

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:

Body weight, food consumption, mating days, sexual cycle test value (number of estrus), organ weight, gestation period, number of corpora lutea, number of implantation marks, number of pups born - average value and standard deviation were calculated for each group. First, the homogeneity of dispersion among the substance administration groups was tested by the Bartlett method. When the variances were uniform, the Dunnett multiple comparison test was used to compare with the control group, and when the variances were not uniform, the Steei multiple comparison test was used to compare with the control group.
The copulation rate, fertility rate, birth rate and sex ratio of offspring are determined by the χ 2 test, and the stillbirth rate, birth rate and 4-day survival rate determined by the Wilcoxon rank sum test. The control group and each treatment group were compared. In all cases, significance level was set to 5%. The measured values for the offspring were processed in litter units.

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):

In males in the 300 mg/kg group, salivation was observed from 36 days after administration. Approximately 9 cases were observed. Recovery had occurred within 30 minutes after dose administration.
In addition, hair loss was noted in individual males and females in the control group and in 1 female in the 100 mg/kg group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 300 mg/kg group died immediately after administration on Day 4 immediately after dosing as did one female in the 30 mg/kg group on Day 13 after dosing. In the 300 mg/kg group, 1 female showed decreased activity, slow breathing, pale skin, swelling of the abdomen, and dirt in the lower abdomen after 17 days of administration. The animal was humanely killed. Necropsy/tissue histopathology was performed.
At necropsy of these deaths, dark redness of the lungs, constriction of the lungs, foamy fluid retention in the trachea, black-red spots of the oesophagus. Fat droplets, which were considered to be corn oil, were found in the alveoli, it was concluded that the cause of death was the error in administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no difference in body weight between male and female in each group compared with the control group throughout the administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Relative to controls, food intake was statistically significantly increased in males in the 30 mg/kg group on Day 49 of administration.

Haematological findings:

no effects observed

Description (incidence and severity):

No difference was observed between each treatment group and the control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males of the 300 mg/kg group, a statistically significant decrease in total cholesterol and urea nitrogen and an increase in triglyceride were observed. A statistically significant decrease in the A/G ratio was observed in the 100 mg/kg group. These were all mild changes.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, animals of the 300 mg/kg group showed hyperplasia of the squamous epithelium of the forestomach and granulomatous inflammation of the submucosa. Vacuolarization of the squamous epithelium and oedema of the squamous subepithelial tissue from the squamous epithelium were observed in 10 cases, and hyperplasia of the squamous epithelium of the forestomach was observed in 1 animal of the 100 mg/kg group. In addition, erosion of the integument was found on the skin in 1 male control group with hair loss.
In females, hyperplasia of the anterior gastric forestomach epithelium, granulomatous inflammation of the submucosa was seen in 7 animals and erosion in 2 animals of the 300 mg/kg group. In addition, atrophy of the thymus was observed in one animal of the 300 mg/kg group. However, similar changes were observed in one animal of the control group.
One of the 2 animals of the 100 mg/kg group autopsied because all of the offspring died had one lung. Cellular infiltration mainly of neutrophils and atrophy of the thymus were observed, but no abnormality was observed in the other case.
In the case of death of the male in the 300 mg/kg group due to administration error, the common changes were oedema, neutrophils, and histiocytes from the squamous epithelium of the forestomach to the submucosa. In addition to the presence of infiltrates, pulmonary haemorrhage and oedema were observed in the one deceased male, and haemorrhage of the oesophageal muscle layer and cellular infiltration mainly of neutrophils in the one female of this group following humane kill from the study. Atrophy of the thymus and necrosis of the tubular epithelium in one part of the renal cortex were observed. In addition, in one female in the 30 mg/kg group which died due to administration error, congestion of the lungs, edema, aggregation of foam cells, infiltration of neutrophils, lymphocytes, etc., and interstitial fluid in the bronchiole lumen and thymic atrophy was observed.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slight, but statistically significant, reduction in the number of estrus in the 30 and 100 mg/kg groups. Similar changes were not observed in the 300 mg/kg group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

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, but there was a tendency for decrease of developmental parameters (total number of pups born, delivery index and live birth index).

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effects were observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

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%

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) 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.

No effects on the oestrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices were observed to indicate an effect on reproductiver performance. 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 was considered to be 300mg/kg/day for reproductive performance of parents and for development of offspring.