Formaldehyde, polymer with benzenamine, hydrogenated

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

other: 28 day repeat dose

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4-bis[(4-aminocyclohexyl)methyl]aniline; 2,4-bis[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 2-[(1-aminocyclohexyl)methyl]aniline; 4-[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 4-{[4-({4-[(4-aminocyclohexyl)methyl]cyclohexyl}amino)cyclohexyl]methyl}cyclohexan-1-amine

EC Number:

603-894-6

Cas Number:

135108-88-2

Molecular formula:

Exact identification is not feasible

IUPAC Name:

2,4-bis[(4-aminocyclohexyl)methyl]aniline; 2,4-bis[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 2-[(1-aminocyclohexyl)methyl]aniline; 4-[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 4-{[4-({4-[(4-aminocyclohexyl)methyl]cyclohexyl}amino)cyclohexyl]methyl}cyclohexan-1-amine

Test material form:

liquid: viscous

Details on test material:

Formaldehyde, polymer with benzenamine, hydrogenated of Air Products, Batch No.: AW301903M3

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A total of 38 male and 38 female healthy CD rats of Sprague-Dawley origin were received from Charles River (UK) Ltd., Margate, Kent, England on 27 September 1995.
- Age at study initiation: The rats were 28 _+ 1 days old
- Weight at study initiation: weight range of 68 to 87 g on arrival
- Housing: All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with mesh floors.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: thirteen day acclimatisation period was allowed between delivery of the animals and start of treatmen

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22 °C
- Humidity (%): 34 to 62 % RH
- Air changes (per hr): 19
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

The test substance was administered by oral gavage to rats of Groups 3 to 5 inclusive using a syringe
and rubber catheter at a dose volume of 10 ml/kg/day.
Control animals received the vehicle, PEG, or distilled water at the same dose volume
(10 ml/kg/day).
Each animal received a constant dosage based on its most recently recorded bodyweight. Animals
were treated once daily.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

MPCA was administered by oral gavage, once daily to three groups of rats for a minimum of twenty eight
consecutive days, at dosage levels of 15, 150 or 300 mg/kg/day . The test material was prepared
as suspensions in PEG (50% w/v aqueous polyethylene glycol 300) at concentrations of 1.5, 15 or
30 mg/ml and was administered at a dosage volume of 10 ml/kg/day. Vehicle control (Group 2)
received the vehicle (PEG) alone at the same dose volume (5 ml/kg/day) and a further group of
control animals (Group 1) received distilled water at 5 ml/kglday.
All rats of Groups 1, 3 and 4 (distilled water, 15 and 150 mg/kg/lday respectively) and five males and
five females from Group 2 and five males and four females of Group 5 (vehicle control and
500 mg/kg/day respectively) were killed following the four-week treatment period (Day 32, 10
November 1995). The remaining animals (five males and five females from Group 2 and two males
and four females of Group 5) were retained for a two-week recovery period, following which, they
were also killed (Day 46, 24 November 1995).
Bodyweights, food consumption and clinical observations were recorded during the study. Blood and
urine samples were taken from all rats shortly prior to termination following the four week treatment
and two-week recovery periods. All animals were killed and shortly examined macroscopically
specified tissues were then prepared for histopathological examination

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

5 males and 5 females per group

Control animals:

yes

Details on study design:

The study was designed to assess the systemic toxicity of MPCA to the rat when repeatedly
administered orally for a period of 4 weeks. Additional animals from the control and high dosage
groups were retained for a 2 week post-treatment observation period.
The albino rat was chosen as the test species as it has been shown to be a suitable model for this type
of study and is the species recommended in the test guidelines. The strain of rat used was chosen on
account of the availability of background data.
The rats were dosed orally as the test substance may be ingested accidentally.
The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity
investigation performed at this laboratory (Report number AIP 401960687). The high level dosage
of 300 mglkg was considered to be the highest dosage that could be tolerated for twenty-eight days
of dosing. The low and intermediate dose levels were selected on the basis of the key dosages
relative to EEC labelling requirement.

A total of 38 male and 38 female healthy CD rats of Sprague-Dawley origin were received from Charles River (UK) Ltd., Margate, Kent, England on 27 September 1995.
The rats were 28 +/- 1 days old, in a weight range of 68 to 87 g on arrival. A thirteen day
acclimatisation period was allowed between delivery of the animals and start of treatment.
All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with
wire mesh floors.
A standard pelleted laboratory rodent diet (Special Diet Services Rat and Mouse Maintenance Diet)
and drinking water were provided ad libitum.
The batches of diet used for the study were analysed for nutrients, possible contaminants and microorganisms.
Results of the routine physical and chemical examination of drinking water at source, as conducted,
usually weekly by the supplier, were made available to Huntingdon Life Sciences Ltd. as quarterly
summaries.
The rats were housed in Building R17 Room 5. Animal room temperature was controlled in the range
19 to 22°C and relative humidity was controlled in the range 34 to 62% RH. These parameters were
continuously monitored using a Kent Clearspan MI05 7day chart recorder. Air exchange was
maintained at approximately 19 air changes per hour and lighting was controlled to provide 12 hours
artificial light (0700-1900 hours) in each 24-hour period.
The health status of all animals was monitored, by daily observation throughout the acclimatisation
period, to ensure that the rats selected for final assignment to the study were satisfactory.
Four days prior to the start of treatment each animal was weighed and seventy rats were randomly
allocated to five groups, two groups consisting of ten males and ten females and three groups
consisting of five males and five females. This allocation was carried out using a computer program,
so that the weight distribution within each group was similar and the initial group mean bodyweights
were approximately equalised.
Each rat was identified within each cage by ear-punch and individually by tail mark (tattoo).
Following the commencement of treatment spare animals were removed from the study. No further
investigations were performed on these animals.
The cages (each containing five rats) constituting each group were distributed in batteries in such'a
manner that possible environmental influences arising from their spatial distribution were equilibrated,
as far as possible, for all treatment.

MPCA was administered by oral gavage, once daily to three groups of rats for a minimum of twentyeight
consecutive days, at dosage levels of 15, 150 or 300 mg/kg/day . The test material was prepared
as suspensions in PEG (50% w/w aqueous polyethylene glycol 300) at concentrations of 1.5, 15 or
30 mglrnl and was administered at a dosage volume of 10 rnllkglday. Vehicle control (Group 2)
received the vehicle (PEG) alone at the same dose volume (5 mllkglday) and a further group of
control animals (Group 1) received distilled water at 5 rnllkglday.
All rats of Groups 1, 3 and 4 (distilled water, 15 and 150 mglkglday respectively) and five males and
five females from Group 2 and five males and four females of Group 5 (vehicle control and
500 mglkglday respectively) were killed following the four-week treatment period (Day 32, 10
November 1995). The remaining animals (five males and five females from Group 2 and two males
and four females of Group 5) were retained for a two-week recovery period, following which, they
were also killed (Day 46, 24 November 1995).
Bodyweights, food consumption and clinical observations were recorded during the study. Blood and
urine samples were taken from all rats shortly prior to termination following the four week treatment
and two-week recovery periods. All animals were killed and shortly examined macroscopically
specified tissues were then prepared for histopathological examiation.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Three male and two female rats from the high dosage group died during the treatment period. For high dosage rats, clinical signs included hunched posture, paddling of forepaws, unsteady gait, thin appearance, lethargy, soft faeces, wet urogenital region, distended stomach, partially closed eyes, noisy respiration and ungroomed appearance during the treatment period. Ungroomed appearance persisted for the first 5 days of the recovery period following which no toxicologically important clinical signs were noted.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three male and two female rats from the high dosage group died during the treatment period. For high dosage rats, clinical signs included hunched posture, paddling of forepaws, unsteady gait, thin appearance, lethargy, soft faeces, wet urogenital region, distended stomach, partially closed eyes, noisy respiration and ungroomed appearance during the treatment period. Ungroomed appearance persisted for the first 5 days of the recovery period following which no toxicologically important clinical signs were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gains were markedly lower than controls for male and females rats from the high dosage group during the treatment period. Gains were also lower for female rats of the intermediate dosage group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was lower than control for high dosage group males during the treatment period. During the recovery period, food consumption was similar for control and treated rats.

Food efficiency:

no effects observed

Description (incidence and severity):

Food consumption was lower than control for high dosage group males during the treatment period. During the recovery period, food consumption was similar for control and treated rats.

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

no effects observed

Description (incidence and severity):

Water consumption for high dosage group females was higher than control during Week 3. During the recovery period, water consumption was similar for control and treated rats.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no other findings for other parameters measured, including haematology that were considered to be related to treatment with MPCA.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At the end of the treatment period higher glutamic-pyruvic transaminase (GPT) and glutarnicoxaloacetic transaminase (GOT) levels were recorded for high-dosage group rats. GOT levels were also slightly higher for intermediate dosage females. Urea nitrogen

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Lower urinary volumes for high dosage group rats and higher specific gravity was recorded for male rats of the high dosage groups, and also the intermediate dosage group, at the end of the treatment period. Higher protein levels

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the 2-week recovery period, adrenal weights were higher than controls for high dosage group males.

Gross pathological findings:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS
For high dosage rats, clinical signs included hunched posture, paddling of forepaws, unsteady gait, thin appearance, lethargy, soft faeces, wet urogenital region, distended stomach, partially closed eyes, noisy respiration and ungroomed appearance during the treatment period. Ungroomed appearance persisted for the first 5 days of the recovery period following which no toxicologically important clinical signs were noted.

MORTALITY
Three male and two female rats from the high dosage group died during the treatment period. Deaths
occurred from Day 8 to Day 30. Clinical signs seen among these animals prior to death included
ungroomed appearance, hunched posture, paddling of forepaws, unsteady gait, lethargy, soft faeces
and fur loss. Macroscopic examination revealed watery distension of the gastrointestinal tract, general
ungroomed appearance and brown stained fur. No treatment-related findings were seen at
microscopic examination. Due to the incidence and distribution of the deaths, and the clinical and
macroscopic observations, the deaths were considered to be treatment-related.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gains were markedly lower than controls for male and females rats from the high dosage
group during the treatment period. Gains were also lower for female rats of the intermediate dosage
group. The gains recorded for high dosage group rats during the recovery period were satisfactory.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was lower than control for high dosage group males during the treatment period.
During the recovery period, food consumption was similar for control and treated rats.

FOOD EFFICIENCY

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption for high dosage group females was higher than control during Week 3. During
the recovery period, water consumption was similar for control and treated rats.

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY

CLINICAL CHEMISTRY
At the end of the treatment period higher glutamic-pyruvic transaminase (GPT) and glutarnicoxaloacetic
transaminase (GOT) levels were recorded for high-dosage group rats. GOT levels were
also slightly higher for intermediate dosage females. Urea nitrogen levels were higher and cholesterol
levels lower for high dosage females and triglyceride levels were higher for intermediate and high
dosage females. There were no treatment-related effects at the end of the recovery period.

URINALYSIS
Lower urinary volumes were recorded for high dosage group rats and consequently higher specific
gravity was recorded for male rats of the high dosage groups, and also the intermediate dosage group,
at the end of the treatment period. Higher protein levels were recorded for female rats.
At the end of the recovery period, high specific gravity and protein levels and low pH were recorded
for high dosage group male rats.

ORGAN WEIGHTS
Liver weights (bodyweight adjusted) were higher than control for intermediate and high dosage male
rats at the end of the treatment period. Adrenal weights were higher than control for high dosage
group males and females and kidney weights were higher than control for intermediate and high
dosage group male and female rats.
At the end of the 2-week recovery period, adrenal weights were higher than controls for high dosage
group males.

GROSS PATHOLOGY
At termination, pale and mottled kidneys, minimal adipose tissue, stained and badly groomed fur were
noted for the majority of high dosage group rats and watery contents of the small intestine was seen
for the high dosage males.
Following the 2 week recovery period, pale kidneys were noted for one high dosage group female
rat and badly groomed fur was noted for one high dosage group male rat. Fur loss was noted for one
male and all female rats for the high dosage group.

Treatment-related findings were reported in the kidneys, adrenals, liver and spleen.
Kidneys - Vacuolatiodfine vacuolation of the cortical tubular epithelium in male and female rats
receiving 150 or 300 mg/kg/day and in recovery male and female rats.
Basophilia and vacuolation of the cortical tubular epithelium and eosinophilic casts within cortical
tubules in male rats receiving 300 mglkglday and tubular dilation in male rats receiving 150 and
300 mg/kg/day. The basophilia and vacuolation and the tubular dilation were present in recovery
male rats.
Adrenals - Diffuse cortical hypertrophy in male and female rats receiving 300 mg/kg/day and in
recovery male and female rats.
Liver - Centrilobular hepatoctye enlargement in male rats receiving 150 or 300 mg/kg/day and in
recovery male rats.
Spleen - Lymphocytolysis was reported in occasional male rats receiving 150 or 300 mg/kg/day and
female rats receiving 300 mg/kg/day. It was present in recovery male rats. This findings is
considered of minor toxicological importance.

OTHER FINDINGS
There were no other findings for other parameters measured, including haematology that were
considered to be related to treatment with MPCA.
The only findings of note for control animals receiving the vehicle, PEG 300 in comparison to
distilled water controls were lower bodyweight gains for females, higher water consumption for
males, higher urinary specific gravity (males and females), lower urinary volume (females) and lower
urinary pH (males). All other differences from vehicle control rats in comparison to distilled water
controls were considered incidental. There findings were not considered to have affected the
evaluation of findings related to treatment with MPCA.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

The treatment-related effects seen at the high and intermediate dosage groups, in particular in the kidney, were considered to be adverse and represent the potential of the test substance to cause serious damage to health. According to Commission Directive 93/21/EEC the R48 (harmful) risk
phase is required for Formaldehyde, polymer with benzenamine, hydrogenated (MPCA).
There were no treatment-related effects at the low dosage of 15 mg/kg/day and this dosage therefore represents the no-observed-effect level (NOEL) in the rat for MPCA when administered orally for at least 28 days.

Executive summary:

The treatment-related effects seen at the high and intermediate dosage groups, in particular in the kidney, were considered to be adverse and represent the potential of the test substance to cause serious damage to health. According to Commission Directive 93/21/EEC the R48 (harmful) risk

phase is required for MPCA (Formaldehyde, polymer with benzenamine, hydrogenated).

There were no treatment-related effects at the low dosage of 15 mg/kg/day and this dosage therefore represents the no-observed-effect level (NOEL) in the rat for MPCA when administered orally for at least 28 days.