m-phenylenebis(methylamine)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-12-28 to 1999-03-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2001

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

temperature or humidity of the animal room deviated from the set range on two days during the study period. However, each deviation was slight and for a short time, and was judged not to have affected the integrity of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

m-phenylenebis(methylamine)

EC Number:

216-032-5

EC Name:

m-phenylenebis(methylamine)

Cas Number:

1477-55-0

Molecular formula:

C8H12N2

IUPAC Name:

1-[3-(aminomethyl)phenyl]methanamine

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): m-xylylenediamine (MXDA)
- Molecular weight: 136.11
- Physical state: Colourless liquid
- Storage condition of test material: Bottled and plugged tightly, stored in test substance storeroom, protected from sunlight

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nippon Charles River Co Ltd (Atsugi-shi, Kanagawa)
- Age at study initiation: 10 weeks
- Weight at study initiation: 354-399 g (males); 216-247 g (females)
- Housing: Barrier sustained animal room (width 5.7 x depth 10.0 x height 2.5 m). Animals were housed individually in a cage with aluminium front and stainless steel mesh floor (width 15.8 x depth 23.8 x height 16.0 cm). Cage specifications were varied during the mating period and, from day 18 of pregnancy, dams were housed individually in a cage with an aluminium front and stainless steel mesh floor (width 36.8 x depth 25.0 x height 16.0 cm) with a lactation tray and nursery material. Cages were exchanged every other week and feeding trays were exchanged weekly.
- Diet: Free access to NMF pellet diet (sterilized by radiation) manufactured by Oriental Industries (Chuo-ku, Tokyo) administered via a nozzle
- Water: Free access to tap water administered via a nozzle
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 24 ± 3 degrees Centigrade
- Humidity: 55 ± 20 degrees Centigrade
- Air changes: 15 per hour
- Illuminance (lux): 150-300
- Photoperiod: 12 hours per day (07:00 to 19:00)

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: Official purified water (Lot 1051)

Details on exposure:

- Pre-treatment observation: 2 weeks before administration began
- Confirmation of childbirth was conducted at 09:00 to 10:00 and day 0 of lactation applied accordingly.
- Post-treatment observation: 1 day for adult animals. Offspring were observed until day 4 of lactation.

Details on mating procedure:

Females whose vaginal smear was examined for 14 days before mating were housed with males of the same group on a 1:1 basis for a maximum of 5 days. Copulation was judged to be successful by confirming the presence of sperms in the vaginal smear the next morning, designated day 0 of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was dissolved in official purified water (Lot 1051 obtained from Kyouei Pharmaceutical Co Ltd) and adjusted to make 5, 15 and 45 mg/L solutions for each group. After preparation, the solution was kept in the dark under room temperature until use. In addition, the test substance solution at concentrations of 1 and 60 mg/L were confirmed in a pilot study to be stable for 8 days at room temperature after preparation.

The solution for each dosing group was analyzed before the start of administration. The results showed that concentrations were between 99.2 and 106.9 % of indicated concentration. Therefore it was confirmed that each dose solution contained the prescribed quantity of 1,3-benzenedimethanamine. Further analysis, conducted by the manufacturer after the administration period, showed the purity was 99.8% and confirmed that the test substance was stable during the administration period.

Duration of treatment / exposure:

- Males: Duration of administration was 48 consecutive days (14 days before mating, 14 days during mating and 20 days after the end of the mating period.
- Females: Duration of exposure was 14 days before mating, during the mating period (maximum 5 days), throughout the gestation period after copulation and 3 days of lactation after parturation (41-45 days). In females which did not deliver after copulation, administration was completed for 40 days until the day before necropsy.
- Offspring: Not dosed

Frequency of treatment:

Seven days per week

Details on study schedule:

Animals were classified according to their body weight and allocated to each group by random selection. Twelve males and 12 females per group were prepared. All females were observed for estrus cycle for 8 days before allocation, and those animals which had normal estrus cycle were used for allocation. Identification of animals after group allocation was done by tattooing an individual number in the ear auricle and by attaching a card with animal ID number on each cage.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
12 males and 12 females

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
12 males and 12 females

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150mg/kg/day
Basis:
actual ingested
12 males and 12 females

Doses / concentrations 4

Remarks:

Doses / Concentrations:
450mg/kg/day
Basis:
actual ingested
12 males and 12 females

No. of animals per sex per dose:

Twelve animals of each sex per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

In a 28-day repeated dose toxicity study, post administration signs such as salivation, decrease in locomotion activity, piloerection, and abdominal distention were seen in males and females of the 600 mg/kg group, and decrease in food intake and supression of body weight gain were seen in males of the 600 mg/kg group. One male and 4 females out of 12 rats in the group died. In addition, ulceration and epithelial hyperplasia associated with hyperkeratosis were seen in the fore-stomach, and myeloid hyperplasia in the bone marrow, hypertrophy and vacuolation of adrenal cortical cells, and dilation of the cecum were seen in males and females of this group. Based on these results, a two week pilot study (number 4211) was conducted using the same doses as those in the 28-day repeated dose toxicity study (0, 10, 40, 150 and 600 mg/kg). During the pilot study, 2 out of 6 males and 1 out of 6 females died in the 600 mg/kg group. Salivation in males and females, suppression of body weight gain in males, and increase in adrenal weight were also observed in this group after administration of the test substance.

Since male and female animals died in the 600 mg/kg group and planned administration was three times longer than in the pilot study, 450 mg/kg was selected as the highest dose, and 150 and 50 mg/kg, dividing by a common ratio of 3, were applied.

Enforced oral administration was used and the administration volume was set as 1 mL per 100 g body weight. The exact volume was calculated using the latest body weight available from measurements taken during pre-mating and mating periods in males and females. In addition, during the lactation period, volume was calculated based on body weight obtained on day zero of lactation. Enforced oral administration was conducted once daily on seven days per week using a stomach cannula. Purified water of official grade was similarly administered to the control group.

Positive control:

No data

Examinations

Parental animals: Observations and examinations:

All male and female animals were observed daily during the study period.

Body weight
Males were weighed on days 1 (start of study) 8, 15, 22, 29, 36, 43 and 49 (necropsy day). The body weight gain from days 1 to 49 was calculated. Females were weighed on days 1 (start of study), 8 and 15, and the body weight gain from day 1 to day 49 was calculated. In addition, females with successful copulation were weighed on days 0, 7, 14 and 21 of gestation, and females after parturition were weighed on days 0 and 4 of lactation. Body weight gains from day 0 to day 21 of gestation and from day 0 to day 4 of lactation were calculated.

Food intake
For males, the food was weighed on days 1 (start of study), 8, 15, 22, 29, 36, 43, and 48 (the day before autopsy), and the mean intake was calculated from intake between a certain day and the next measuring day. In addition, cummulative food intakes from day 1 to day 15 and from day 22 to day 48 were calculated. In females, the food was weighed on days 1 (start of study), 8 and 15 and the mean daily food intake was calculated from intake between a certain day and the next measuring day. In addition, cummulative food intake from day 1 to 15 was calculated. In females with successful copulation, food was weighed on days 0, 7, 14 and 21 of gestation. In females with parturition, food was weighed on days 0 and 4, and mean daily food intake was calculated from intake between a certain day and the next measuring day. In addition, cummulative food intake from day 0 to day 21 of gestation was calculated. Food intake of cohabiting animals was not measured during the mating period.

Oestrous cyclicity (parental animals):

Observation of estrus cycle was continued until the day of successful copulation. The number of days between one estrus and the next were taken as duration of estrus cycle, and the mean duration of estrus cycle was calculated. In addition, the incidence [(number of animals with abnormal estrus cycle/number of animals observed)/100] of abnormal estrus cycle (estrus cycle other than 4 or 5 days) was calculated.

Sperm parameters (parental animals):

No data

Litter observations:

Observations were taken daily for gestation period and evidence of delivery.

Postmortem examinations (parental animals):

Organs examined at necropsy (macroscopic and microscopic)
- Organ weights in males: Thymus, adrenal, testes and epididymides
- Organ weights in females: Thymus, adrenal
- Histopathology: Thymus, stomach, adrenal, ovaries, uterus, vagina, testes, epididymides, abnormal site of spleen from two animals, nasal wall from one animal, small intestine from three animals, large intestine from five animals, seminal vesicle from one animal, liver from one animal and lung from one animal.

Other examinations
- Inspected at necropsy: Appearance of the oral cavity, nostril, cranial cavity, skeleton, external appearance and dissected face of brain and spinal cord, thoracic cavity, abdominal cavity and pelvic cavity with viscera, cervial tissues and organs. All abnormalities noted during gross examination were noted together with location, size, hardness and other pertinent details.

Postmortem examinations (offspring):

No histopathology was carried out on offspring

Statistics:

A multiple comparison test was applied to data on body weight, food intake, number of corpora lutea, implantation site, number of offspring, number of offspring dead, sex ratio, mean length of estrus cycle, duration of pregnancy, implantation rate, delivery rate, birth rate, ratio of external abnormalities, ratio of offspring alive on day 4 after birth, organ weight, and organ weight/body weight ratio (relative organ weight).

Firstly homogeneity of variance was analysed by Bartlett's test. When the variance was homogenous, significant differences between the control group and each treated groupwere analysed by Dunnett's multi-comparison test. When variance was heterogenous, significant differences between the control group and each treated group were analysed by Steel's test. For comparison of birth rate, copulation index and fertility index, a chi square test was used. The incidence of abnormal estrus cycle and the incidence of pathology findings were analysed by Fisher's direct probability test. Those histopathological findings which increased in severity in the treated groups were ranked and then analysed by Mann-Whitney's U test. In addition, for the result concerning live offspring during the lactation period, a mean value per one dam was used as one sample. Statistical analysis at 5 % and 1 % significance were performed by a two-tail test.

Reproductive indices:

Copulation index [(number of animals with successful copulation/number of mated animals) x 100] was calculated. A female which died on day 2 of pregnancy was excluded from the summing up of fertility index because it was impossible to judge whether that female was prenant. A male was examined only on histopathology when considering males who failed to impregnate but was included in summing up when considering males who succeeded in impregnation.

Offspring viability indices:

No data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

General observations

In the groups treated with 150 mg/kg or more, salivation and nasal noise was seen in male animals. Ulceration of the fore-stomach and adhesion to surrounding tissue was seen in males from the 450 mg/kg group.

In the 450 mg/kg group, salivation and nasal noise were seen in female animals. In addition, suppression of bodyweight gain, reduced food intake, and ulceration of the fore-stomach were observed during necropsy of females.

Toxic response/effects by dose level

Death and general signs

One male animal from the 150 mg/kg group died on Day 38. Three further males from the 450 mg/kg group died on Days 31, 38 and 39. One female in the 450 mg/kg group died on day 2 of pregnancy.

Dose dependent salivation was the most common observation and was noted in males and females from the 150 mg/kg and 450 mg/kg groups. Salivation observed in a few animals in weeks 5 and 6 and in half the animals in week 7 of administration was a transient change, which appeared immediately after dosing and disappeared 30 minutes later. In males of the 450 mg/kg group, salivation was observed in around half of the animals and most of the animals in week two or later. In week 1, salivation appeared immediately after administration and disappeared after 30 minutes. However, the effect lasted longer as administration continued, being continuously observed at 90 to 180 minutes after administration during the latter stages of the study. In one female of the 150 mg/kg group, salivation was seen on day 2 of pregnancy, with the same transience as observed in males. In females of the 450 mg/kg group, salivation was observed in most animals at pre-mating and mating stages plus the early stages of pregnancy. A gradual decrease in the number of animals exhibiting salivation was found in the middle to late stages of pregnancy. Only one animal showed salivation during the lactation period after delivery. In a few females from the 450 mg/kg group, salivation appeared immediately after administration and lasted for 60 minutes during the gestation period. During other periods, salivation appeared immediately after administration and disappeared 30 minutes later.

In addition, the following observations were made: nasal noise (1 male, 150 mg/kg group and 3 males and females, 450 mg/kg group), irregular respiration (1 male, 150 mg/kg group), abdominal distension, emaciation, staining of fur, piloerection, hypothermia (1 male, 150 mg/kg group and 1 female, 450 mg/kg group), nasal discharge (1 male and 1 female, 450 mg/kg group), pale appearance (1 male, 150 mg/kg group and 2 females, 450 mg/kg group), ptosis (1 male, 150 mg/kg group plus 2 males and 1 female, 450 mg/kg group), prone position (1 female, 450 mg/kg group).

Spontaneous changes such as eye discharge (1 male, control group) and alopecia (1 female, 150 mg/kg group plus 1 male and 1 female, 450 mg/kg group) were also observed.

Bodyweight

Bodyweights of males from the 450 mg/kg group were smaller than those of the control group on Day 8 and thereafter. Bodyweight gain (62 ± 20 g) was also significantly less than the control group (129 ± 26 g).

Females from the 450 mg/kg group had bodyweights on day 14 and 21 of pregnancy that were less than the control group. Bodyweight gain during gestation (140 ± 19 g) was also significantly less than the control group (170 ± 31 g).

Food intake

Food intake in the 450 mg/kg dose group was less than that of the control group and the result was statistically significant. In males, the daily intake between Days 1 to 8, Days 29 to 36, and Days 43 to 48 was smaller. The cumulative intake between Days 1 to 15 was also smaller. The daily intake of female animals between days 1 to 8 and, cumulatively, between Days 1 to 15 was also smaller.

Organ weights

In males from the 450 mg/kg group, absolute weight of the thymus decreased (190 ± 60 mg) compared with controls (277 ± 73 mg). Absolute (90 ± 14 mg) and relative weight (20.526 ± 3.256 %) of the adrenal increased compared with control values of 63 ± 9 mg and 12.382 ± 1.925 %. The relative weight of the testes also increased (0.741 ± 0.029 % compared with 0.0667 ± 0.047 % for the control animals). The relative weight of the thymus was inclined to decrease although no statistical difference was observed (45.090 ± 12.432 % compared with 54.228 ± 13.221 % for the controls).

Necropsy

In males, ulceration of the fore-stomach was seen in 9 animals of the 450 mg/kg group and the incidence was increased with statistical significance in comparison to the control group. Of these 9 animals, six had adhesion of the stomach with surrounding tissue. Other changes were observed in both the control and the test groups.

Females who underwent natural delivery showed ulceration of the fore-stomach (9 animals, 450 mg/kg group) and atrophy of the thymus (6 animals, 450 mg/kg group) and these effects were statistically significant. Three females from the 150 mg/kg group had fore-stomach ulceration with stomach adhesion to surrounding tissues, 3 had mucosal-thickening of the fore-stomach and 1 animal had atrophy of the spleen. Other changes were observed in both the control and test groups.

One female from the control group failed to deliver and a dead foetus was found in the uterus on day 25 of pregnancy.

Histopathology

Male animals showed ulceration of the fore-stomach, squamous epithelium hyperplasia with hyperkeratosis in the fore-stomach and diffuse hyperplasia in the adrenal cortex with statistically higher significance compared to the control group. For ulceration in the fore-stomach, incidence of severe cases also increased with statistical significance compared to control animals.

Females who underwent natural delivery showed ulceration of the fore-stomach, squamous epithelium hyperplasia with hyperkeratosis in the fore-stomach and diffuse hyperplasis in the adrenal cortex. The incidence of these effects was higher than the control group and was statistically significant. As with the male animals, incidence of severity of ulceration increased with statistical significance compared to controls.

Copulation and fertility

The female from the 450 mg/kg group that died on day 2 of pregnancy was excluded from conception observations. All other females conceived. No intergroup differences were observed in the mean duration of the oestrus cycle. No intergroup differences were seen in the incidence of abnormal oestrus cycle.

Delivery and lactation

No abnormalities were seen in delivery. The values of pregnant period, number of corpora lutea, number of implantation sites, number of offspringand number of offspring born alive were similar among each group. No differences among groups were seen in gestation index, implantation index, birth index, sex ratio or viability index on day 4.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Details on results (P0)

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Details on results'

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

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:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

Details on results (F1)

Offspring morphology

Bodyweight and necropsy findings showed dwarfism (bodyweight less than 60 % of mean bodyweight of control group) in only one offspring from the 450 mg/kg dose group. No intergroup differences were observed in bodyweight on day 0 and day 4. Offspring were necropsied on day 4 of lactation and a thymus remnant was seen in 5 males of the control group, 3 males and 1 female of the 50 mg/kg group, and 2 males of the 450 mg/kg group. Other sporadic changes observed in males were whitish spotting, dark spotting, adhesion with diaphragm, yellowish change and anomalous nodule in the liver, pelvic dilation of the kidney, displacement of the kidney, and reddening of the eyeball. In addition, skin bruising was seen in 3 males and females of the smae litter from the 150 mg/kg group.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: see 'Details on results (F1)'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The test substance administration influenced the death of males in the 150 mg/kg group and males and females of the 450 mg/kg group.

Clinical observations were also considered to be caused by substance administration. It was suspected that the substance also caused stimulation of mucus secretion and caused respiratory dysfunction. Other observations such as ptosis and fur staining were considered to be induced by exacerbation of a general condition but not by the substance. Salivation was considered to be an incidental occurance unrelated to the administration of the test substance.

Bodyweight gain was suppressed by substance administration and food intake was also influenced.

The action of the substance appeared to be stronger in males than females. It was therefore concluded that the action of the test substance was sex related.

The substance did not cause any changes in the reproductive organs. The changes in the digestive system were considered to be induced by the corrosive nature of the substance. The substance did not affect any part of the reproductive cycle.

Applicant's summary and conclusion

Conclusions:

No influences on fertility or the next generation were observed