Registration Dossier

Administrative data

Description of key information

Oral:
A NOAEL of 100 mg/kg bw/day was determined in a combined repeated dose/reproduction toxicity study (OECD 422) in rats on methyltetrahydrophthalic anhydride (THPA), this based on changes in clinical chemistry parameters, changes in organ weights and inflammation of the forestomach mucosa.
Dermal and inhalation:
In accordance with REACH Regulation 1907/2006 (Annex IX, Column 2), assessment of repeated dose toxicity by dermal or inhalation exposure is not required as data are available for the (default) the oral route.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995.12.6-1997.6.5
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
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
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 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Various studies have been undertaken on tetrahydromethylphthalic anhydride (MTHPA). This is a structural analogue of 3 -MTHPA D4 in which the methyl substitution is not defined or fixed in a specific position on the carbon cyclic structure, as opposed to 3 -MTHPA D4 where the methyl substitution is fixed at the 3C position. 3 -MTHPA D4 is regarded as a specific isomer of MTHPA.

Oral:

In the key study (Izumi, 1997), 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.

No 90 day toxicity study with tetrahydromethylphthalic anhydride (MTHPA) is available. Taken all data from MTHPA and structural analogue substances together, a new 90 day toxicity study with MTHPA is not required and not in line with concerns regarding animal welfare and the use of animals for experimental purposes. The data available for chemically almost identical substances in different species and for exposure periods of 90 days support the findings noted in OECD 422 study taking the time extrapolation factor into account. Therefore, the OECD 422 study is considered to represent a reliable basis for DNEL derivation for MTHPA.

Dermal and inhalation:

In accordance with REACH Regulation 1907/2006 (Annex IX Column 2) assessment of repeated dose toxicity by dermal or inhalation exposure is not required as data are available for the (default) oral route. The most relevant exposure route of this substance to investigate systemic toxic effects is regarded as oral. Inhalation or dermal exposure gives rise to local effects (sensitising and irritating effects) which occur before systemic toxicity becomes relevant.


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

On the basis of the data available, classification of the tetrahydromethylphthalic anhydride isomer 3 -MTHPA D4 is not justified according to the criteria given in Directive 67/548/EEC (CLP) or Regulation 1272/2008 (GHS).