1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Immunotoxicity

Description of key information

OPPTS 870.7800, subacute, rat, oral: NOAELtoxicity = 31.0 mg/kg bw/day; LOAELtoxicity = 334.2 mg/kg bw/day; NOAELimmunotoxicity ≥ 737.9 mg/kg bw/day (females)

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

immunotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Deviations:

yes

Remarks:

Flow cytometric analyses of splenic sub-populations not performed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar (Hsd Cpb:WU)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males 145 g mean (126-161 g); females 141 g mean (124-159 g)
- Fasting period before study: not applicable
- Housing: Groups with 2 or 3 animals in Makrolon cages Type IV on low dust wood granules (Ssniff Spezialdiaeten Inc. Soest/Westfalen). For environmental enrichment wooden blocks suppied by Tapvei OY, 73620 Kortteinen, Finland, were provided to each cage and renewed as necessary. Wood granules and wooden blocks were randomly analysed for contaminants.
- Diet: Provimi Kliba 3883 G4 S25, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 2008 To: 19 March 2008

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance was mixed in the diet at the appropriate concentrations at room temperature weekly and maximally used for the stability period (15 days) proven by analytical investigation.
- Mixing appropriate amounts with (Type of food): Provimi Kliba 3883 G4 S25
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test substance in the diet were checked prior to study start and once during the study. This analytical investigation showed the test substance to be homogeneously distributed and stable in the concentration range used beyond the period of use, and that the test substance content agreed with the target concentrations.

Actual concentrations in the diets fed during the study were within the range of 107 to 110% of the nominal concentrations. After 15 days at room temperature test substance contents in specially prepared test diet formulations containing 100 and 10000 ppm of test substance ranged from 103 to 104% of nominal concentrations.

Duration of treatment / exposure:

males 29 days, females 30 days

Frequency of treatment:

continuously in the diet

Dose / conc.:

300 ppm (nominal)

Remarks:

corresponding to 31.0 mg/kg bw/day for females and 27.7 mg/kg bw/day for males

Dose / conc.:

3 000 ppm (nominal)

Remarks:

corresponding to 334.2 mg/kg bw/day for females and 258.8 mg/kg bw/day for males

Dose / conc.:

6 000 ppm (nominal)

Remarks:

corresponding to 737.9 mg/kg bw/day for females

Remarks:

high-dose of males reduced after 16 days due to marked effects; corresponding to 528.0 mg/kg bw/day for males

No. of animals per sex per dose:

8

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: As requested by the sponsor.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays) for morbidity and mortality; once daily for general clinical examinations
- Cage side observations included: morbidity, mortality, general clinical examinations

DETAILED CLINICAL OBSERVATIONS: Yes, with open field observations
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as mean daily test substance intake per animal and per kg bw from the consumption and body weight data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all rats of the study including all unscheduledly deceased animals; organs and tissues were subjected to thorough gross pathological examination. Localisation, size, colour and consistency were described. Spleen and/or thymus were preserved and archived.

Cell viabilities:

SPLEEN: Yes
- Method: Trypan Blue exclusion
- Dose groups: all
- No. of animals: all

Humoral immunity examinations:

ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: Yes
- Method: Plaque Forming Cell Assay
- Dose groups: all
- No. of animals: all

Other examinations:

Organ Weights: Spleen, thymus

Positive control:

none

Statistics:

Statistical evaluations on body and organ weight were done using the Dunnett-test in connection with variance analysis. All variables that were not dichotomous were described by sex, dose group and time point using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum). For the statistics of samples drawn from continuously distributed random variables three types of statistical tests were used, the choice of the test being a function of prior knowledge obtained from former studies. Provided that the variables in question were approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared more likely, a p-value adjusted Welch test was applied. If the vidence based on experience with historical data indicated that the assumptions for parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate. In order to avoid false-positive statements also biological and toxicological relevance was considered.

Immunotoxicity values from treated groups were compared with those from the control group by the Mann Whitney or the Wilcoxon significance test (Rank Sum Test or One Way ANOVA or Kruskal-Wallis ANOVA) at significance levels of 5% (two tailed). Before these evaluations, data are proven for homogeneity of variance according to Cochran.
As a result of the multiple tests the overall probability of error is considerably greater than the p value suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation of statistical significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

mortality observed, treatment-related

Description (incidence):

adverse

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

adverse

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

not adverse

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

secondary effect

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical changes were observed within the daily in-cage or weekly detailed clinical observations in males up to 3000 ppm and in females at 300 ppm.
High-dose rats revealed piloerection, hunched back, uncoordinated gait, high stepping gait and tremor; one male showed additionally pallor, sunken flanks, poor general condition and reduced motility.
One 3000 ppm female showed tremor during the detailed clinical observation in the last week of the study.

Survival was not affected by the treatment with the test substance in males up to 3000 ppm and in females up to 6000 ppm. One high-dose male had to be killed in moribund condition an day 12 of the study.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was unaffected in males at 300 ppm and in females up to 3000 ppm. High dose males showed clear body weight loss; on day 15 the difference to the start of the study was 15% and the difference to mean control was 48%. Because of this significant body weight reduction the concentration of this group was reduced to 6000 ppm after 2 weeks of treatment. After reducing the dose the body weight increased immediately. However, at the end of the treatment the difference to control was still 39%.

Males at 3000 ppm showed a slight trend to retarded body weight development. The differences to control were about 8% during the study (p>0.05).

Females at 6000 ppm revealed slightly but statistically significantly retarded body weight development (about 11-14% lower than control).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was slightly decreased in males at 3000 ppm (18% per animal and 11% per kg body weight) and clearly decreased at high dose concentrations (51% per animal and 22% per kg body weight).

Females at 3000 and 6000 ppm showed increased food intake (up to 30%).

Treatment resulted in the following mean daily test substance intake related to body weight (averaged over the study period): 27.7, 258.8 and 528.0 mg/kg bw for males and 31.0, 334.2 and 737.9 mg/kg bw for females. The test substance intake corresponded to the theoretical dose factor for the two lower dose groups. For the highest male dose group the intake was slightly lower compared to the theoretical factor due to the decreased food intake in this group, whereas in high dose females the intake was slightly higher compared to the respective control.

WATER CONSUMPTION
Water intake per animal was reduced in high dose males (30%). Due to the lower body weights at this dose mean body weight related water intake was somewhat increased (about 16%).

In treated females mean body weight related water intake was slightly but not dose-dependently higher than in controls (14%, 6% and 10%). Without dose dependency these findings in females are considered as incidental.

GROSS PATHOLOGY
One high dose male which was killed in moribund condition showed emaciation and diminished spleen and thymus at necropsy.

The absolute mean weights of spleen and thymus were decreased in high dose males. Since the relative weights were comparable to controls these changes were attributable to the decreased body weights in this group.

CELL VIABILITIES
Splenic cell counts showed a statistically significant decrease in males at the highest dose (count: 204.4±68.8 X 10e6). This decrease was, however, within the range of the normal variance of this parameter (mean of historical data is 460 X 10e6 ± 37% SD), and thus in itself of no biological relevance. Although, a consequence due to the reduced general health condition cannot be excluded.

In females the splenic counts did not exhibit any test substance induced effect or a statistically significant difference between test substance-treated and control females up to and including the highest dose level.
Therefore, splenic cell counts did not show any test substance-induced effects in any dose group compared to the respective control groups.

HUMORAL IMMUNITY EXAMINATIONS
All test substance-treated animals exhibit higher numbers of plaque forming cells than the vehicle treated animals, which showed relatively low numbers in both sexes. These higher values were not be considered of immunotoxicological relevance, since there is no statistical significance and no dose dependency. Furthermore, it has to be mentioned that a slight increase in PFC could also be induced by stress, as indicated by the slight reduction in body weight in treated animals.

Cell viabilities:

effects observed, treatment-related

Description (incidence and severity):

secondary effect

Humoral immunity examinations:

effects observed, treatment-related

Description (incidence and severity):

not biologically relevant

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Other findings:

not examined

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 27.7 mg/kg bw/day in males and 31.0 mg/kg bw/day in females

Dose descriptor:

LOAEL

Remarks:

toxicity

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: corresponding to 258.8 mg/kg bw/day in males and 334.2 mg/kg bw/day in females

Dose descriptor:

NOAEL

Remarks:

immunotoxicity

Effect level:

>= 6 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on immunotoxicity observed

Remarks on result:

other: corresponding to 528.0 mg/kg bw/day in males and 737.9 mg/kg bw/day in females

Determination of cell counts in the spleen and performance of the Plaque Forming Assay gave no evidence for treatment-related effects after subacute treatment with the test substance. Therefore, the NOAEL for immunotoxicity for both gender was set to the highest concentrations tested, 6000 ppm for females or 10000/6000 ppm for males. These concentrations corresponded to a mean daily intake of 528.0 mg/kg bw/day in males and 737.9 mg/kg bw/day in females. Thus, no hints for immunotoxic effects of the test substance were determined.

The overall NOAEL was 300 ppm, corresponding to a mean daily intake of 27.7 mg/kg bw/day for males and 31.0 mg/kg bw/day for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

737.9 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on immunotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is data available from a subacute oral immunotoxicity study in rats which was conducted according to the EPA guideline OPPTS 870.7800 under GLP-conditions. In this study the females were exposed to dietary concentrations of 300, 3000 and 6000 ppm (corresponding to 31.0, 334.2 and 737.9 mg/kg bw/day) and the males to 300, 3000 and 10000/6000 ppm (the dietary concentration of high-dose males was reduced to 6000 ppm after 16 days due to marked effects; the concentrations corresponded to 27.7, 258.8 and 528.0 mg/kg bw/day, averaged over the study period); males were exposed for 29 days, females for 30 days, one day later the animals were sacrificed. Five days prior to sacrifice humoral immune reactions against sheep red blood cells (SRBC) were induced by intravenous injection of 10e8 SRBCs. In addition to the usual examinations in subacute animal studies, special attention was paid to the lymphatic organs spleen and thymus. Both were preserved after sacrifice, their organ weights were determined, the viability of the spleen cells was determined, and the humoral immune response to sheep red blood cells (SRBC) was assessed in a Plaque Forming Cell (PFC) Assay with splenic lymphocytes. The splenocytes were mixed with SRBCs, and the amount of SRBC-specific B cells among the splenocytes was detected on glass slides after incubation with guinea pig complement. The SRBC-specific antibodies produced by the activated B cells will lyse the SRBCs in their surrounding by addition of compliment; the clear plaques forming around the B cells are visible under the microscope.

One high dose male had to be killed in moribund condition on day 12. There were clear body weight loss and clearly decreased food and water consumption observed in this group, therefore the dietary concentration was reduced to 6000 ppm. High-dose rats showed clear signs of toxicity like piloerection, hunched back, uncoordinated gait, tremors. Males at 3000 ppm and females at 300 ppm remained unaffected; one female at 3000 ppm showed tremor in the last week of the study, therefore the overall NOAEL was determined to be 300 ppm, corresponding to 31.0 mg/kg bw/day in females. The overall LOAEL was set to 3000 ppm (334.2 mg/kg bw/day in females). Determination of spleen cell counts and the PFC-Assay gave no indication for treatment-related effects after subacute treatment with the test substance. Therefore, the NOAEL for immunotoxicity for both gender was set to be greater than 6000 ppm (737.9 mg/kg bw/day) for females and 10000/6000 ppm (528.0 mg/kg bw/day) for males.

Justification for classification or non-classification

The test substance did not exert any immunotoxic effect on rats after subacute exposure.

According to the criteria of Regulation (EC) No 1272/2008 the test substance does not have to be classified for exerting immunotoxic effects after repeated exposure.