Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Structural similarities of the target substance to the source substances include the (bi)cyclic ring structure with a carboxylic acid anhydride group as the single reactive moiety. For the target substance the bicyclic ring structures contains a double bond at a specific location within the ring and also contains a substituted methyl group at a specific location on the ring structure whereas for the source substance neither the position of the double bond nor the methyl group are specified.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a specific isomer of the source substance, tetrahydromethylphthalic anhydride (MTHPA), in which neither the location of the double bond nor the methyl substitution are defined. MTHPA, has a stated composition comprising the target substance 1,2,3,6-tetrahydro-3-methylphthalic anhydride (CAS No. 5333-84-6, EC No. 226-247-6) together with tetrahydro-4-methylphthalic anhydride (CAS No. 34090-76-1, EC No. 251-823-9), 1,2,3,6-tetrahydro-4-methylphthalic anhydride (CAS No. 3425-89-6, CAS No. 222-323-8) and 3-cyclohexene-1,2-dicarboxylic anhydride, 4-methyl- (CAS No. 19438-64-3).


3. ANALOGUE APPROACH JUSTIFICATION
Generally, the physical and chemical properties do not show major differences. All the above mentioned cyclic anhydrides have comparable boiling temperatures, low vapour pressure and, from a physiological point of view, similar partition coefficients. Differences in melting point may be explained by the fact that the substances are representing multi-constituent or mono-constituent substances.
The cyclic anhydrides rapidly hydrolyse in contact with water and the methyl substituted cyclic anhydrides appear to show a low potential for biodegradation. Based on their physico-chemical properties the substances are expected to have low bioaccumulation potentials and comparable adsorption/desorption properties. The dicarboxylic acid degradation product arising from hydrolysis is the moiety of concern with respect to effects in the aquatic environment. The substances have a rather a low potential to cause toxicity to water based species including fish, daphnia, algae and microorganisms.
Acute toxicity is low. All of the substances are expected to be highly irritant to the eye and be both skin and respiratory sensitisers and a harmonised classification is in place for these properties (Index No. 607-240-000). The absence of a mutagenic potential has been demonstrated in various guideline in-vitro tests. The outcome of the available mammalian studies conducted by the oral route suggest local effects (irritation of the stomach mucosa) probably arising from pH effects of the di-acid degradation product to be significant.
Due to structural similarities, comparable physical/chemical properties such as the molecular weight, partition coefficient and vapour pressure, the toxicokinetic profile of the registered substance and the potential structural analogue substances are also expected to be comparable in terms of physiological absorption, distribution, metabolism and excretion processes. The carboxylic acid anhydride group which rapidly hydrolyses to form the di-acid once in contact with physiological liquid, mainly determine the fate of the substance within the body. Available toxicokinetic data demonstrate that cyclic anhydrides are commonly metabolised to the corresponding di-carboxylic acids within the body and are finally excreted in urine.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
read-across source
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), of which 3 -MTHPA D4 is an isomer, 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a combined repeated dose toxicity/reproduction and developmental toxicity study (Izumi, 1997) tetrahydromethylphthalic anhydride (MTHPA), of which 3 -MTHPA D4 is an isomer, 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 were observed on the oestrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices to indicate an effect on reproductive performance. Examination at delivery and during the lactation period revealed no 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 is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring.

A "higher tier" study such as a 2 -generation study was waived for the following reasons: The available data for structural homologues of MTHPA indicate neither the potential for teratogenic effects nor for reproduction toxicity in different species. These data, together with the available information on MTHPA, allow a scientific validated evaluation of the respective endpoints and further tests would not be in line with concerns regarding animal welfare and the use of animals for experimental purposes.


Short description of key information:
In a combined repeated dose toxicity/reproduction and developmental toxicity study with rats on tetrahydromethylphthalic anhydride (MTHPA), of which 3-MTHPA D4 is an isomer, the NOAEL is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring.

Effects on developmental toxicity

Description of key information
In a combined repeated dose toxicity/reproduction and developmental toxicity study with rats, the NOAEL is considered to be 300 mg/kg/day for reproductive performance of parents and for development of offspring. The available data for structural homologues of tetrahydromethylphthalic anhydride (MTHPA) indicate neither potential for teratogenic effects nor for reproduction toxicity in different species. These data, together with the available information on MTHPA, allow a scientific validated evaluation of the respective endpoints and further tests would not be in line with concerns regarding animal welfare and the use of animals for experimental purposes.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A developmental toxicity study was waived for the following reasons:

The available data for structural homologues of MTHPA indicate neither the potential for teratogenic effects nor for reproduction toxicity in different species. These data, together with the available information on MTHPA, allow a scientific validated evaluation of the respective endpoints and further tests would not be in line with concerns regarding animal welfare and the use of animals for experimental purposes.

The available data from an OECD 422 screening study are regarded as having supporting value only as only basic parameters for developmental toxicity were examined. In this, groups of Crj:CD(SD) rats (12/sex) received, by gavage dose levels of 0 (Vehicle), 30, 100 and 300 mg/kg bw/day MTHPA, of which 3 -MTHPA D4 is an isomer. No external anomalies were apparent. The NOAEL was considered to be 300 mg/kg/day for development of offspring.

Justification for classification or non-classification

Based on the available information methyltetrahydrophthalic anhydride and its isomers does not require classification or labelling according to Directive 677548/EEC (DSD) and to Regulation 1272/2008/EC (CLP).

Additional information