Registration Dossier

Administrative data

Description of key information

The oral administration of diisopropanol-p-toluidine by gavage to male and female Wistar rats for 3 months caused signs of systemic toxicity and mortality at 80 mg/kg bw/d in males during the first 59 days of administration. Systemic toxicity was not observed in males from study 60 onwards administrating 40 mg/kg bw/d and not in females tested up to 20 mg/kg bw/d. The lowest observed adverse effect level (LOAEL) in males was 80 mg/kg bw/d. Since only adverse findings had been observed under the conditions of the present study in clinical observation during the first 59 days, the no observed adverse effect level (NOAEL) was 40 mg/kg bw/d for male and at least 20 mg/kg bw/d for female Wistar rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2017 - July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test substance No.: 11/0416-3
Batch identification: 33468624U0
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
Age when supplied: 29 ± 1 days
Age at the start of administration period: 42 ± 1 days

The animals were housed together (5 animals per cage) in H-Temp polysulfonate cages type 2000P (floor area about 2065 cm2). FOB and motor activity measurements were conducted in polycarbonate cages type III (floor area about 800 cm2). The cages and wire covers were supplied by TECNIPLAST, Hohenpeissenberg, Germany resp. by Ehret, Emmendingen, Germany. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Lignocel Block Large) supplied by Rettenmaier & Söhne GmbH Co KG, Rosenberg, Germany and large play tunnels (Art. 14153) supplied by PLEXX B.V., Elst, Netherlands were added for environmental enrichment. The animals were accommodated in fully air-conditioned rooms in which central air conditioning resulted in uniform temperature range of 20-24°C, a range of relative humidity of 30-70% and 15 air changes per hour. The day/night cycle was 12 hours (12 hours light from 06.00 h -18.00 h, 12 hours dark from 18.00 h-06.00 h). Deviations from these ranges did not occur.

The animal room was completely disinfected prior to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week with water containing an appropriate disinfectant.

The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum.
Route of administration:
oral: gavage
Details on route of administration:
The administration volume was 4 mL/kg body weight.
Vehicle:
corn oil
Details on oral exposure:
Diisopropanol-p-toluidin was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume, subsequently mixed with a magnetic stirrer. At room temperature the test-substance preparations formed not applicable suspensions. To reach applicable forms of the test-substance preparations the preparations were heated at 50°C and stirred occasionally for minimum half an hour before application. After this treatment the test-substance preparations formed solutions. The test-substance preparations were produced at least weekly and stored at room temperature. The administration volume was 4 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The various analyses confirmed
• the stability of the test-substance preparations for a period of 7 days at room temperature including heating/shaking periods at 50°C for 4 hours,
• the correctness of the prepared concentrations.

The analyses of the test-substance preparations were carried out at the Analytical ChemistryLaboratory of Experimental Toxicology and Ecology of BASF SE , Ludwigshafen, Germany.
The stability of Diisopropanol-p-toluidin in Corn oil Ph.Eur. 8.0 at room temperature for a period of 7 days including heating/shaking periods at 50°C for 4 hours was proven during the start of the administration period.
Concentration control analyses of test-substance preparations were performed at the beginning and towards the end of the administration period in all concentrations. All further samples were not analyzed and stored frozen (at -20°C).
Duration of treatment / exposure:
3 months (90 days)
Frequency of treatment:
The test substance was administered daily by gavage for 3 months. Control animals received only the vehicle.
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
male:
The dose of male animals of the test group 3 was reduced from 80 mg/kg bw/d to 40 mg/kg bw/d from study day 60 onwards after two males were found dead and observing severe findings (like general poor conditions, smeared fur, abdominal position, and convulsions) in at least 5 of 10 male animals.
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
male & female
Dose / conc.:
7 mg/kg bw/day (actual dose received)
Remarks:
male & female
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
Remarks:
female
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels of this study were selected based on the findings in two preceding studies. In an OECD 422 study 2.5, 10 and 40 mg/kg bw/d were investigated. Thereby, tremors in 9 of 10 female Wistar rats were observed and three females found dead on the first day of administration of 40 mg/kg bw/d Diisopropanol-p-toluidin. No test substancerelated adverse findings were observed in male animals at the highest dose tested. In a 14 days repeated dose test study only slight effects on food consumption and body weight change were observed at 100 mg/kg bw/d in male rats.
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS

Mortality
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.

Clinical observations
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

Detailed clinical observations
Detailed clinical observations (DCO) 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.

Food consumption
Food consumption was determined weekly (as representative value over 7 days) for each cage. The average food consumption per cage was used to estimate the mean food consumption in grams per animal and day.

Water consumption
Drinking water consumption was observed by daily visual inspection of the water bottles for any changes in volume.

Body weight data
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 study 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 study day 0 was calculated as body weight change.

Functional observational battery
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting in the morning. At least one hour before the start of the FOB the animals were transferred to single-animal cages. Drinking water was provided ad libitum, but 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 sensory motor tests as well as reflex tests.

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during the examinations in order not to influence the behavior of the rats. Attention was paid to:

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes.

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity assessment
Motor activity (MA) was also measured in the early afternoon onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over
12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

Ophthalmoscopy
The eyes of all animals were examined prior to the start of the administration period.

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer). In the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided). Urine was sampled overnight. On the following day, the samples were examined in a randomized sequence (the list of randomization instructions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The following parameters of the animals were examined:
- hematology
- clinical chemistry
- urin analysis

Pathology:
- necropsy
- organ weights
- organ/tissue fixation
- histopathology
Sacrifice and pathology:
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Statistics:
see attached background material
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of systemic toxicity were observed in males of test group 3 (80 mg/kg bw/d) during the first 59 day of administration. At this lethal dose, following clinical observations had been observed: poor general condition and abdominal position in 8 of 10 males, tonic-clonic convulsions and smeared fur in the mouth regions in 5 of 10 males, tonic convulsions and encrusted nose, respirations sounds and labored respirations, piloerection in 2 of 10 males as well as clonic convulsions, slight unsteady gait, slight tremors, red smeared fur in nose region, redding of skin in hind limb region, and pale skin in single animals. These findings were observed as isolated or continuing findings on single or several days starting on day 12 of administration. After reducing the dose in test group 3 from 80 mg/kg bw/d to 40 mg/kg bw/d for males on study day 60, no adverse clinical findings were observed. No further clinical findings were observed in male animals of test groups 2 and 1 (20 and 7 mg/kg bw/d) and in females up to 20 mg/kg bw/d, the highest dose tested in females, during the entire administration period.
The pattern of clinical findings in males at 80 mg/kg bw/d indicated partially neurotoxicity potential of the test substance, however, neither the functional observation battery and the motor activity measurement nor the pathological assessment determined further adverse findings supporting this. No histopathological alterations of neural structures were observed. Since two male rats died at 80 mg/kg bw/d after 12 days or 56 days of repeated administration, the clinical pattern could not be excluded but it also cannot be dedicated absolutely certain to be caused by either a neurotoxic potential or mortality.

Please see also attached summary document.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two animals of test group 3 (80/40 mg/kg bw/d) were found dead in this study. Male No. 36 was found dead on stud day 12 and male No. 31 on study day 56 within 2 hours after administration of the respective day.

Please see also attached summary document.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes of mean body weights and mean body weight change values in both sexes were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related findings. All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Please see also attached summary document.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At study day 92, in males of test group 3 (80/40 mg/kg bw/d) total protein levels were significantly higher compared to controls. The mean was marginally above the historical control range, whereas those of albumin and globulins in this test group were within historical control ranges (males, total protein 60.64-64.93 g/L, albumin 33.28-39.61 g/L, globulins 22.72-30.69 g/L). Therefore, the increased total protein levels in males of test group 3 were regarded as incidental and not treatment-related.

Please see also attached summary document.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.

At the end of the in-life phase in the urine sediment of males of test group 3 (80/40 mg/kg bw/d), incidences of granular and epithelial cell casts were increased. These higher incidences were not found in females. This sex-specific, isolated finding in male rats of this age is most probably due to alpha-2u-globulinuria. This effect is not relevant for humans (Hard et al., 1993).

Please see also attached summary document.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery:
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:
Home cage observations: No test-substance related adverse effects were observed.
Open field observations: No test-substance related adverse effects were observed.
During open field observation in male animal No. 16 of test group 1 (7 mg/kg bw/d) opacity in and exophthalmos of the left eye were observed. These single findings in the low dose group were assessed as spontaneous in nature and not treatment related.

Sensorimotor tests/ reflexes: No test-substance related effects were observed.
Quantitative parameters: No test-substance related effects were observed.

Motor activity measurement:
In males of test group 3 (80/40 mg/kg bw/d) motor activity’s single interval No. 8 was increased. In females of test group 3 (20 mg/kg bw/d) single interval No. 11 was decreased. Additionally, single interval No. 11 was decreased in females of test group 2 (7 mg/kg bw/d). Furthermore, single interval No. 8 was increased in male animals of test group 1 (7 mg/kg bw/d). All findings occurred without a dose-depending effect and were assessed as incidental. Thus, regarding the overall motor activity as well as single intervals, no test-substance related deviations were noted for male and female animals of all test groups.

Please see also attached summary document.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with control group 0 (=100%), kidney (114%) and liver (112%) absolute weights were significantly increased in test group 3 male animals. All other mean absolute weight parameters in males and all mean absolute weight parameters
in females did not show significant differences when compared to the control group 0.

When compared with control group 0 (=100%), the liver mean relative organ weights were significantly increased in test groups 1 (107%) and 3 (108 %) of male animals.

Please see also attached summary document.

Both mean absolute (2.446g) and relative (0.594%) kidney weights of test group 3 males were within historical controls (absolute: 2.075g- 2.57g, relative: 0.564 – 0.652%). The mean absolute kidney weights of the concurrent control (2.149 g) were within the historical control range, while the mean relative kidney weights (0.544%), was slightly below the historical control range. As both mean absolute and relative kidney weights of test group 3 were within historical controls while the concurrent control group showed a relative weight below the historical range, the increased mean absolute kidney weight of male animals of test group 3 were assessed as not treatment-related. Furthermore, the relative kidney weight in test group 3 males was not statistically significantly changed.

The mean absolute liver weight of test group 3 of the present study (9.066 g) was slightly higher than historical controls (7.611 – 8.95 g). The mean relative liver weight of test group 3 (2.203%) and test group 1 (2.181%) were within historical controls (2.11 – 2.302%). The mean relative weight of the liver of the concurrent control group (2.045%) was, however, below historical controls. Additionally, no histopathologic findings were noted in treated male animals. The increased mean absolute weights of test group 3 males and the increased mean relative liver weights of test group 1 and 3 males were therefore assessed as incidental and not treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings were single or few observations. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Decedents: There were no macroscopic findings to explain the death of male animals No. 31 and 36.

Please see also attached summary document.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No histopathological alterations of neural structures were observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

This includes the occurrence of eosinophilic droplets in the proximal tubules in the kidneys which showed an immunohistochemical staining pattern positive for alpha2u protein similar to that described by Cesta et al 2013 in animals 6 and 7 of the control group and animal 31 and 32 of test group 3. No further findings consistent with alpha2u nephropathy were observed. No differences in incidence between test group 3 and the control were noted, the severity grading was comparable, therefore this finding was assessed as not treatment-related. Necrosis/fibrosis in the heart was also assessed as incidental and not treatment-related as incidences and grading were comparable between all test groups. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Decedents
There were no histological findings to explain the death of male animals No. 31 and 36, both of test group 3. Both showed atrophied mandibular glands and one animal small seminal vesicles / coagulating glands. This was assessed as a sign of poor body condition of these two animals prior to death.

Please see also attached summary document.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
behaviour (functional findings)
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no substance-related adverse effects were observed
Remarks on result:
other: highest dose tested
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatemend-related, advers effects were observed
Remarks on result:
other: highest dose tested
Critical effects observed:
yes
Lowest effective dose / conc.:
80 other: mg/kg bw/d (actual dose received) male
System:
other: general poor conditions, smeared fur, abdominal position, convulsion, mortality
Organ:
not specified
Treatment related:
yes
Critical effects observed:
no
Lowest effective dose / conc.:
40 other: mg/kg bw/d (actual dose received) male
Critical effects observed:
no
Lowest effective dose / conc.:
20 other: mg/kg bw/d (actual dose received) male & female
Critical effects observed:
no
Lowest effective dose / conc.:
7 other: mg/kg bw/d (actual dose received) male & female
Critical effects observed:
no
Lowest effective dose / conc.:
2.5 other: mg/kg bw/d (actual dose received) female

Dose group: 80/40 mg/kg bw/d in males (dose in males reduced from study day 60 onwards)

Clinical Examinations up to study day 59 (80 mg/kg bw/d)

• 2/10 males found dead (No. 31 on study day 56, No. 36 on study day 12)

• slight and moderate poor general condition in 8/10 males

• posture in abdominal position in 8/10 males

• moderate tonic-clonic convulsions in 5/10 males (incl. No. 31 on study day 56)

• slight clonic convulsions in 1/10 males

• tonic convulsions in 2/10 males (incl. No. 31 on study day 56)

• slight unsteady gait in 1/10 males

• slight tremors in 1/10 males

• red smeared fur in mouth region in 5/10 males

• red smeared fur in nose region in 1/10 males (No. 36 on study day 12)

• red bloody discharged left eye in 1/10 males (No. 31 on study day 56)

• moderate labored respiration in 2/10 males (incl. No. 31 on study day 56)

• encrusted nose in 2/10 males

• respiration sounds in 2/10 males (incl. No. 31 on study day 56)

• piloerection in 2/10 males (incl. No. 31 on study day 56)

• reddening of skin in hindlimb in 1/10 males (No. 31 on study day 56)

• pale skin at entire body in 1/10 males

Pathology up to study day 59 (80 mg/kg bw/d)

• No macroscopic or histopathologic findings in male animals No. 31 and 36 (found dead)

Clinical Examinations from study day 60 onwards (40 mg/kg bw/d)

• No substance-related, adverse effects were observed.

Clinical Pathology

• No substance-related, adverse effects were observed.

Pathology

• No substance-related, adverse effects were observed.

Dose group: 20 mg/kg bw/d in males and females

Clinical Examinations, Clinical Pathology, and Pathology

• No treatment-related, adverse effects were observed.

Dose group: 7 mg/kg bw/d in males and females

Clinical Examinations, Clinical Pathology, and Pathology

• No treatment-related, adverse effects were observed.

Dose group: 2.5 mg/kg bw/d in females

Clinical Examinations, Clinical Pathology, and Pathology

• No treatment-related, adverse effects were observed.

Conclusions:
The oral administration of Diisopropanol-p-toluidin by gavage to male and female Wistar rats for 3 months caused signs of systemic toxicity manifested in general poor conditions, smeared fur, abdominal position, convulsions and mortality at 80 mg/kg bw/d in males during the first 59 days of administration. Systemic toxicity was not observed in males from study 60 onwards administrating 40 mg/kg bw/d and not in females tested up to 20 mg/kg bw/d. The lowest observed adverse effect level (LOAEL) in males was 80 mg/kg bw/d. Since only adverse findings had been observed under the conditions of the present study in clinical observation during the first 59 days, the no observed adverse effect level (NOAEL) was 40 mg/kg bw/d for male and at least 20 mg/kg bw/d for female Wistar rats. Thus, the overall NOAEL for this study is 20 mg/kg bw/d.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline study

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

Repeated dose toxicity: oral route

key study:

The oral administration of diisopropanol-p-toluidine by gavage to male and female Wistar rats for 3 months (BASF SE, 2018, OECD 408) caused signs of systemic toxicity manifested in general poor conditions, smeared fur, abdominal position, convulsions and mortality at 80 mg/kg bw/d in males during the first 59 days of administration. Systemic toxicity was not observed in males from study 60 onwards administrating 40 mg/kg bw/d and not in females tested up to 20 mg/kg bw/d. The lowest observed adverse effect level (LOAEL) in males was 80 mg/kg bw/d. Since only adverse findings had been observed under the conditions of the present study in clinical observation during the first 59 days, the no observed adverse effect level (NOAEL) was 40 mg/kg bw/d for male and at least 20 mg/kg bw/d for female Wistar rats.

supporting information:

In a repeated dose oral toxicity study (BASF SE, 2013, OECD 422) diisopropanol-p-toluidine was administered to Wistar rats (4 groups of 10 male and 10 female animals each) via oral gavage at dose levels of 0, 2.5, 10, 40 mg/kg bw/day (males) and 20 [40 on pre-mating day 0] mg/kg bw/day (females). The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females. No mortality and no clinical signs were observed for male animals. Nine females of the high dose group (40 mg/kg bw/d) showed severe tremors on premating day 1 and three female animals were found dead on premating day 1. After reduction of the dose level to 20 mg/kg bw in female animals no signs of general systemic toxicity were observed. No treatment-related effects were observed in mean body weight, body weight changes, food and water consumption of exposed animals throughout the study. Neurological testing did not indicate any neurotoxic potential of the test item. No treatment related changes among hematological, clinical chemistry and urinalysis parameters were observed. When compared to the control (set up 100%), the mean absolute liver weights were significantly increased in male animals of the 2.5 and 40 mg/kg bw/d dose group. Absolute liver weights of male animals did not show a dose response, there were no significant changes in relative liver weights, and a histopathological correlate was not detected, therefore increased absolute liver weights were regarded as incidental. All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group. At gross lesions and histopathology examination all findings were considered to be incidental or were biologically equally distributed over control and treatment groups.

In conclusion, based on mortality and tremors noted at 40 mg/kg bw/d, the No Observed Adverse Effect Level (NOAEL) for females was established at 20 mg/kg bw/day. As no systemic toxicity was observed in males the NOAEL was established at 40 mg/kg bw/d.

Justification for classification or non-classification

Based on the results obtained from repeated dose toxicity studies (OECD 422 & 408), the test substance diisopropanol-p-toluidine is not considered to be subject to classification and labelling for repeated dose toxicity according to Regulation (EC) No 1272/2008 (CLP).