3(or 4)-methylbenzene-1,2-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully reported guideline study to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3(or 4)-methylbenzene-1,2-diamine

EC Number:

248-145-0

EC Name:

3(or 4)-methylbenzene-1,2-diamine

Cas Number:

26966-75-6

Molecular formula:

C7H10N2

IUPAC Name:

3(or 4)-methylbenzene-1,2-diamine

Details on test material:

3,4-TDA: 59.2 g/100 g
2,3-TDA: 35.2 g/100 g
2,4-TDA: 2.01 g/100 g

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were housed together (5 animals per cage) in H-Temp (PSU) cages (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel FS 14 fibres, dust free bedding, Motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen (floor area about 800 cm2) and small amounts of absorbent material (Type Lignocel FS 14 fibres, dust free bedding, ). For enrichment wooden gnawing blocks (Typ NGM E-022), were added. The animals were accommodated in fully air-conditioned rooms in which central air conditioning
guaranteed a range of temperature of 20-24 °C, a range of relative humidity of 30 – 70% and 10 air changes per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours (12 hours light from 6.00 – 18.00 h, 12 hours dark from 18.00 – 6.00 h). Deviations from these ranges did not occur. The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal. Food and drinking water (from water bottles) were available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: acidified drinking water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water for a period of 4 hours was proven before the start of the administration period (Study No.: 01Y0575/078010). Homogeneity and concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start, the mid and at the end of the administration period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 50, 250 mg/kg BW / day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. At the start of the administration period (day 0) the rats were 42 +1 days old. The test substance was administered daily for about 28 days. Control animals received the vehicle only. All animals were sacrificed after a fasting period (withdrawal of food) of at least 16 hours.

Examinations

Observations and examinations performed and frequency:

A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily before and after the administration for any clinically abnormal signs. Abnormalities and changes were documented for each animal. Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. For these observations the animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high).

Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.
Individual drinking water consumption was determined weekly over a period of 4 days and calculated as mean water consumption in grams per animal and day. Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 h. At least one hour before the start of the FOB the animals were transferred singly to Polycarbonate cages. Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Motor activity measurements were carried out in all animals towards the end of the administration period. The examinations were performed using the TSE Labmaster System. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. 18 beams were
allocated per cage. The numbers of beam interrupts were counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out from 14:00 h onwards. On account of the measuring variant "staggered", the starting time was vary by the time which was needed to place the animals in the cages. For each animal, the measurement started individually when the 1st beam was interrupted. The end of data acquisition was exactly 1 hour later. The animals were without food or water during the period of motor activity measurement. After the transfer of the last animal in each case, the room of measurement was darkened. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.


Prior to the start of the administration period and on day 28 the eyes of all animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic.

Sacrifice and pathology:

The animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
Liver, Kidneys, Adrenal glands, Testes, Epididymides, Heart, Ovaries, Uterus, Spleen, Brain, Thymus, Thyroid glands

Organ/Tissue fixation
The following organs/tissues were preserved in neutral buffered 4% formaldehyde solution:
All gross lesions, Salivary glands (mandibular and sublingual glands), Esophagus, Stomach (forestomach and glandular stomach), Duodenum, jejunum and ileum, Cecum, colon and rectum, Liver, Pancreas, Brain, Pituitary gland, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar cords), Eyes, Adrenal glands, Thyroid glands, Parathyroid glands, Trachea, Lungs, Pharynx, Larynx, Nose (nasal cavity), Aorta, Heart, Bone marrow (femur), Lymph nodes (mesenteric and axillary lymph nodes), Spleen, Thymus, Kidneys, Urinary bladder, Testes, Ovaries, Oviducts, uterus and vagina, Epididymides, prostate and seminal vesicle, Female mammary gland, Skin, Skeletal muscle, Sternum with marrow, Femur with knee joint, Extraorbital lacrimal glands. From the liver, each one slices of the lobus dexter lateralis and the Lobus sinister lateralis was fixed in Carnoy’s solution and embedded in paraplast.

Histotechnical processing/Examination by light microscopy and assessment of findings
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings thus:
All gross lesions in all affected animals
All animals in the control and high dose group: Salivary glands (mandibular and sublingual glands), Esophagus, Stomach (forestomach and glandular stomach), Duodenum, jejunum and ileum
Cecum, colon and rectum, Liver, Pancreas, Brain, Pituitary gland, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar cords), Eyes, Adrenal glands, Thyroid glands, Parathyroid glands, Trachea, Lungs, Aorta, Heart, Bone marrow (femur), Lymph nodes (mesenteric and axillary lymph, nodes), Spleen, Thymus, Kidneys, Urinary bladder, Testes Epididymides, Ovaries, Oviducts, uterus and vagina, Prostate, seminal vesicle,
Female mammary gland, Skin.

The hematoxylin-eosin stained slides were assessed by light-microscopy. Gross lesions were correlated with histopathological findings.
The immunorelevant organs and tissues were evaluated according to specific parameters. Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”.

Other examinations:

In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The haematological parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model). Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M;).

Statistics:

Statistics of clinical examinations: Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were performed according to following: Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, footsplay test, motor activity by ‘Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (twosided) for the equal medians.

Body weight, body weight change:A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.

Statistics of clinical pathology: Means, medians and standard deviations of each test group were calculated for several parameters. Further statistical non-parametric analyses were performed.

Organ weights: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Throughout the record, text uses the term "significant" implies that the inter-group differences have attained statistical significance (p < 0.05) when compared with the control group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Based on all endpoints, clinical signs, organ weight, body weight gain, pathology.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mortality:

No animal died prematurely within the study.

Clinical examinations

Eyelid half closure or closure after treatment was observed in all animals of dose group 3 on several days between days 1 and 28. Lower arousal predominately after treatment was observed in all animals of dose group 3 of either sex on several days of the study between days 0 and 27. Piloerection after treatment was observed in 2 male (no. 18 and 19) and 3 female animals (no. 36, 38 and 39) of dose group 3 between study days 7 and 26. Abdominal position after treatment was observed in one female animal (no. 36) of dose group 3 on study days 21, 22 and 23. The above-mentioned findings were assessed as signs of general systemic toxicity and thus related to treatment with the test compound.

Salivation was observed predominantly after treatment in all animals of either sex of dose group 3 (250 mg/kg bw/d) on several days of the study. Additionally, the same finding was observed in 4 female animals (no. 31, 32, 33 and 35) of dose group 2 (50 mg/kg bw/d) on a few days between days 20 and 28. Salivation is often seen in gavage studies and related to the physico-chemical properties of the test compounds in majority of cases. In the present study, the administered test substance preparation was adjusted to pH6 with HCl in order to get a more stabile solution. Thus, the observed salivation is most probably related to a slightly local irritating effect of the test-substance preparation administered by gavage. Therefore, the observed salivation is assessed as related to treatment particularly to the mode of applications but toxicologically irrelevant and thus not adverse. Cornea opacity was observed in one female animal (no. 36) of dose group 3 from day 25 to the end of the study. This single finding was clearly incidental and not related to the test article.

Food consumption

No substance-related effects on food consumption were obtained.

Water consumption

Water consumption was increased in male and female animals of dose group 3 (250 mg/kg bw/d) throughout the entire study up to +36.9% on day 21 in males and +56.3% on day 28 in females. These increased values were most probably caused by a compensatory behaviour due to the above mentioned salivation. Therefore, this finding is clearly substance-related but not an intrinsic adverse effect.

Body weight data

Body weight data were affected only in male animals of the high dose group (group 3, 250 mg/kg bw/d). In particular, body weight was statistically significant decreased in high dose males during the whole study to -11.7% on day 28. The corresponding body weight change was statistically significantly decreased in these animals to -26.3% at the end of the administration period. These body weight data were assessed as related to treatment and clearly a sign of general systemic toxicity and thus adverse.

Food efficiency

Food efficiency was decreased in male animals of dose group 3 throughout the study. Food efficiency is calculated based upon individual values for body weight and food consumption . As only the body weight was decreased , whereas no significant influence on food consumption was measured, the decreased food efficiency values were caused in consequence of an impaired body weight. Therefore this finding is clearly substance-related but not an intrinsic adverse effect.

Functional observational battery (FOB)

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. Besides this, the following examinations were performed during FOB and have to be assessed individually:

Home cage observations: No substance-related findings were observed.

Open field observations: Eyelids closure (slight) was observed in male animal No. 20 and female animal No. 37. In addition female animal No. 36 showed eyelids half closure. All animals are from dose group 3 and these findings are consistent with the same observations described in clinical observations.

Sensorimotor tests/reflexes: A retarded pupillary reflex was observed in two animals of dose group 3 (Nos. 20 and 36). No adaptation of the pupil to light was observed in one female animal of dose group 3 (No. 40).

Quantitative parameters: No substance-related effects were observed.

The above-mentioned findings observed during FOB were assessed as related to treatment with the test substance.

Motor activity measurement

The single intervals number 2, 3 and 6 were statistically significantly decreased in female animals of dose group 3. The overall motor activity, was decreased in males as well as statistically significantly decreased in females of dose group 3.These findings were assessed as substance related.

Ophthalmological examinations

Towards the end of the study on day 28, a final ophthalmological examination was carried out. Within this examination the following findings were observed. The right eye of male animal No. 17 of dose group 3 showed a retina rupture and haemophthalmia. The left eye of one male animal of dose group 3 (No. 16) showed opacity foci. Both findings were observed in single animals only and were therefore assessed as being incidental and thus not adverse. The observed corneal stipplings and remainders of the pupillary membrane were equally distributed between the dosed animals and the controls as well as were without any doseresponse relationship. Therefore, these findings were clearly not related to treatment and without any toxicological relevance.

Absolute weights:

Compared to controls following values (in %) were reached for the dose groups:

..	Male animals			Female animals
	10	50	250	10	50	250
	mg/kg bw	mg/kg bw	mg/kg bw	mg/kg bw	mg/kg bw	mg/kg bw
Terminal body weight	94%	97%	86%**	.	.	.
Thymus	86%	103%	66%**	98%	96%	68%*
Thyroid glands	80%*	88%	78%*	.	.	.

*: p<=0.05 **: p<=0.01

The statistically significant decrease of terminal body weight in 250 mg/kg bw dose groups in male animals is considered a treatment-related effect. It cannot be excluded, that the statistically significant decrease of absolute thymus weights in male and female animals of 250 mg/kg bw dose groups is a treatment-related effect,although a histomorphological correlate is missing.

The decrease of absolute thyroid gland weights in male animals in 10 mg/kg bw as well as 250 mg/kg bw dose group is considered incidental and not related to treatment due to missing dose-response relationship. All other mean absolute weight parameters did not show relevant differences compared to the control group and are regarded to be within the normal biological range of test animals of that age.

Relative Weights

Compared to controls (= 100%) following values (in %) were reached for the dose groups:

..	Male animals			Female animals
	10	50	250	10	50	250
	mg/kg bw	mg/kg bw	mg/kg bw	mg/kg bw	mg/kg bw	mg/kg bw
Liver	103%	110%	121%**	..	..	..
Thymus	91%	107%	75%**	94%	92%	67%**

*: p=0.05 **: p=0.01

The statistically significant increase of relative liver weight in 250 mg/kg bw dose group in male animals is considered a treatment-related effect, although a clear histomorphologic correlate is missing. It cannot be excluded, that the statistically significant decrease of relative thymus weights in male and female animals of 250 mg/kg bw dose groups is a treatment-related effect, although a histomorphological correlate is missing. All other mean relative weight parameters did not show relevant differences compared to the control group and are regarded to be within the normal biological range of test animals of that age.

Pathology:

Gross lesions: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.

Histopathology: All findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.

Applicant's summary and conclusion

Conclusions:

The oral administration of ortho-TDA by gavage over a period of 28 days revealed substance-related adverse findings in animals of both sexes
at 250 mg/kg bw/day. There were general clinical signs of toxicity at this dose and reduced body weight gain. Changes in some in clinical pathology indices indicates the liver as target organ. The no observed adverse effect level (NOAEL) under the conditions of the present study
was 50 mg/kg body weight/day in male and female rats.