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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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
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: 356.3-394.4g, 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
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
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 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
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)
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: 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
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Critical effects observed:
not specified
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
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).
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
Critical effects observed:
no
Reproductive effects observed:
not specified

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%

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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997
Reference Type:
publication
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

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)
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
Vehicle:
other: 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The prepared test solutions were analysed at the start week of administration, and it was confirmed that it they were within the permissible range (± 5%) of the nominal concentration.
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)
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
Duration of test:
Males; for 49 days Females; from 14 days before mating to day 3 of lactation
Doses / concentrationsopen allclose all
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 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: 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

Maternal 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, 17and 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, 17and 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

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
Ovaries and uterine content:
Reproduction parameters females:
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)
Fetal examinations:
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.
Indices:
maternal parameters:
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)

litter parameters:
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)
Historical control data:
no data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the 30 mg/kg group died immediately after dosing on Day 13. 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
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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 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.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
In the 30 and 100 mg / kg groups, the stillbirth rate was statistically significantly increased and the birth rate decreased accordingly. This tendency was observed even in the 300 mg / kg group but was not statistically significant.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No external anomalies were apparent. Thus, the NOAEL is considered to be 300mg/kg/day for development of offspring.

Effect levels (fetuses)

Dose descriptor:
NOEC
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No external anomalies in offspring were apparent. Tus, the NOAEL is considered to be 300mg/kg/day 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 treatment related effects on the oestrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices were observed. Examination at delivery and during the lactation period revealed, no substance related effects 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 wasconsidered to be 300mg/kg/day for reproductive performance of parents and for development of offspring.