Reaction mass of bis[3-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-) and bis[5-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-)

Endpoint summary

Description of key information

Key: the reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 175 mg/kg bw/day for animals of either sex. The study was conducted with a structural analogue substance (other salt type of Basic Red 46).

Supporting: The oral (gavage) administration of Basic Red 46 at dose levels of 250, 500 and 1000 mg/kg bw/day for a period of up to fourteen consecutive days, resulted in treatment related effects in animals of either sex from all treatment groups. The necessary early termination (1000 and 500 mg/kg bw/day), reduction in body weight gain, reduction in food consumption and macroscopic abnormalities detected at all dose levels were sufficient to exclude these dose levels from further investigation. Based on the findings of this study, the Lowest Observed Adverse Effect Level (LOAEL) for Basic Red 46 when administered via oral gavage for 14 days was determined to be 250 mg/kg bw/ day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2017 - 12 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: [yes/no]
- Age at study initiation:
Male: approximately 11 weeks old
Female: approximately 14 weeks old
- Weight at study initiation:
Male: 281 to 343g
Female: 198 to 235g
- Fasting period before study: Not specified
- Housing: All animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

IN-LIFE DATES: From: 25 September 2017 (first day of treatment) To: 27 November 2017 (final day of necropsy).

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Concentration in vehicle (mg/mL): 5, 15, 35
- Dose Level (mg/kg bw/day): 0, 25, 75 and 175.
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples: Distilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples. The concentration of test item in the final solution was quantified by HPLC using UV/Visible detection.

Duration of treatment / exposure:

Approximately six weeks (for males) and up to ten weeks (for females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all treated females.

Sacrifice and pathology:

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Other examinations:

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only).

Statistics:

Where considered appropriate, quantitative data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel
(non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for
these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Throughout the treatment period, noisy respiration, increased salivation, fur stained by the test item (pink/red) and pink stained bedding was evident in animals of either sex from all treatment groups. One male treated with 175 mg/kg bw/day also showed an isolated incidence of hunched posture.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female treated with 175 mg/kg bw/day was found dead on Day 10. This female had mild degenerate/inflammatory changes in the stomach and hyperkeratosis in the stomach, however, the cause of death was not identified. One male treated with 25 mg/kg bw/day was killed in extremis on Day 15 due to the severity of clinical signs noted, which included gasping/labored/noisy respiration, decreased respiratory rate, stained snout, fur stained by test item and a distended abdomen. Changes were apparent in the gastrointestinal tract at necropsy; however, no histopathological changes were evident in the tissues examined. One female treated with 25 mg/kg bw/day was killed in extremis on Day 38 due to difficulties during parturition. There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males from all treatment groups showed a reduction in body weight gain during Weeks 1, 2 and 4, which resulted in a reduction in overall body weight gain. Females treated with 175 mg/kg bw/day showed a reduction in overall body weight gain during maturation. Reductions in body weight gain were also evident during the final two weeks of gestation and during lactation. No adverse effect in body weight development was evident in females treated with 75 or 25 mg/kg bw/day during maturation, gestation or lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males treated with 175 mg/kg bw/day showed a reduction in overall food consumption and food conversion efficiency. No such effects were evident in males treated with 75 or 25 mg/kg bw/day.
Females treated with 175 mg/kg bw/day showed a slight reduction in overall food consumption during maturation and a reduction in food consumption throughout lactation. Food conversion efficiency for these females during maturation was comparable to controls. No such effects on food consumption were evident in females treated with 75 or 25 mg/kg bw/day.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the organ weights measured.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The female treated with 175 mg/kg bw/day that was found dead on Day 10 had a dark liver and red content in the stomach. The stomach was also stained by the test item. The male treated with 25 mg/kg bw/day that was killed in extremis on Day 15 had gaseous distension in the stomach and intestines, small seminal vesicles, a thin cecum, red contents in the stomach and sloughing/thinning of the non-glandular region of the stomach. The female treated with 25 mg/kg bw/day that was killed in extremis on Day 38 had a dead fetus which had rotated in the vagina.
One female treated with 175 mg/kg bw/day had gaseous distension in the cecum, colon and rectum and a further female from this treatment group had raised white areas on the nonglandular region of the stomach. No such effects were evident in males treated with 175 mg/kg bw/day or in animals of either sex treated with 75 or 25 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Stomach: Hyperplasia of the non-glandular mucosa was present in two males treated with 75 mg/kg bw/day and in one male and two females treated with 175 mg/kg bw/day. Hyperkeratosis was also evident in one male each from the 75 and 175 mg/kg bw/day dose group. No such effects were evident in females treated with 75 mg/kg bw/day or in animals of either sex treated with 25 mg/kg bw/day.

Description (incidence and severity):

Thyroid Hormone Analysis
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Details on results:

Other examinations:
Thyroid Hormone Analysis
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Estrous Cycle
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating
There was no effect of treatment on mating performance. With the exception of five animals, all remaining animals mated within four days of pairing.

Fertility
There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Lengths
There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Offspring Litter Size, Sex Ratio and Viability
There was no detrimental effect of treatment with the test item on the mean number of implantations or post-implantation loss at 175, 75 or 25 mg/kg bw/day. Litter size at birth and subsequently during lactation was slightly reduced in females treated with 175 mg/kg bw/day, however, sex ratio and subsequent offspring survival to Day 13 of age was unaffected. No such effect on litter size was evident at 75 or 25 mg/kg bw/day. Sex ratio and subsequent offspring survival to Day 13 of age at 75 and 25 mg/kg bw/day was comparable to controls.

Offspring Growth and Development
As a consequence of the slightly lower litter size at birth in females treated with 175 mg/kg bw/day, litter weights were reduced in these females throughout lactation. Offspring body weight gains in these litters were slightly lower than controls from Day 4 of lactation onwards. No such effects were detected in females treated with 75 or 25 mg/kg bw/day.
Ano-genital distance on Day 1 post partum, visible nipple count in male offspring on Day 13 post partum and clinical signs up to Day 13 of age at 25, 75 and 175 mg/kg bw/day for all treated litters was comparable to controls.

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: local irritant effects in non-glandular stomach resulting in a lower food consumption and lower body weight gain at 75 and 175 mg/kg bw/day

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: local irritant effects in non-glandular stomach resulting in a lower food consumption and lower body weight gain at 175 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed

Critical effects observed:

no

DEVIATIONS FROM STUDY PLAN

The following deviations from the study plan occurred:

Clinical Observations

The one hour post-dosing observations were performed approximately thirty minutes after they were due on Day 30 relative to the start of dosing. All other observations were performed correctly on that day and whilst this omission was regrettable it does not affect the purpose or integrity of the study.

Selection of Animals for Extended Observations

Due to the death of Male No. 29 during the study, Male No. 30 was selected as a replacement for the extended observations. Sensory Reactivity, Motor Activity, Grip Strength, Hematology and Blood Chemistry were all performed on this male, however, at necropsy the full tissue list was not retained in error. In view of the incomplete data for this male, another male (No. 31) had the full extended observations performed. All of the data recorded for both animals will be reported.

Mortalities

There were a small number of instances during the study where the early mortality checks were not performed at times considered acceptable by the Study Director. However, as all animals were monitored twice daily from arrival to termination, this deviation can be considered not to have affected the purpose or scientific integrity of the study.

Litter data

The sex for offspring No. 7 from Litter 94 was not recorded on Day 4 of lactation in error. The sex on Day 1 and Day 13 of lactation for this offspring was confirmed as male, therefore, for reporting purposes, this offspring will be reported as male for Day 4 of lactation.

Conclusions:

‘No Observed Effect Level’ (NOEL) for local toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males. The ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 175 mg/kg bw/day for animals of either sex.

Executive summary:

The oral administration of FAT 31016/T TE to rats by gavage, at dose levels of 25, 75 and 175 mg/kg bw/day was evaluated in this study conducted according to OECD Guideline 422. Clinical signs were detected in animals of either sex from all treatment groups. Episodes of increased salivation and noisy respiration were evident throughout the study. The physical condition of treated males was also affected with reductions in body weight development generally being observed throughout the study. Subsequently, a reduction in overall body weight gain was evident in treated males and reductions in food consumption and food conversion efficiency was also evident throughout the treatment period for males treated with 175 mg/kg bw/day. Females treated with 175 mg/kg bw/day also showed reductions in body weight gains during maturation, the final two weeks of gestation and during lactation and food consumption was reduced in these females during lactation. Observations of this nature are often reported when a test item formulation is unpalatable or irritant and can be associated with gastric irritancy rather than attributable to true systemic toxicity. This was supported microscopically in animals of either sex treated with 175 mg/kg bw/day and in males treated with 75 mg/kg bw/day, where hyperplasia and hyperkeratosis of the non-glandular region of the stomach was evident. Based on the above findings, the ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males.

The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Further the microscopic changes observed were seen in the non-glandular region of the rodent stomach. The corresponding non-glandular area is not present in the man and hence the adverse effects were considered not significant to the man. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 175 mg/kg bw/day for animals of either sex.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

175 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality GLP 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

Oral

The oral administration of the structural analogue substance to rats by gavage, at dose levels of 25, 75 and 175 mg/kg bw/day was evaluated in the study conducted according to OECD Guideline 422. Clinical signs were detected in animals of either sex from all treatment groups. Episodes of increased salivation and noisy respiration were evident throughout the study. The physical condition of treated males was also affected with reductions in body weight development generally being observed throughout the study. Subsequently, a reduction in overall body weight gain was evident in treated males and reductions in food consumption and food conversion efficiency was also evident throughout the treatment period for males treated with 175 mg/kg bw/day. Females treated with 175 mg/kg bw/day also showed reductions in body weight gains during maturation, the final two weeks of gestation and during lactation and food consumption was reduced in these females during lactation. Observations of this nature are often reported when a test item formulation is unpalatable or irritant and can be associated with gastric irritancy rather than attributable to true systemic toxicity. This was supported microscopically in animals of either sex treated with 175 mg/kg bw/day and in males treated with 75 mg/kg bw/day, where hyperplasia and hyperkeratosis of the non-glandular region of the stomach was evident. Based on the above findings, the ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males.

The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Further the microscopic changes observed were seen in the non-glandular region of the rodent stomach. The corresponding non-glandular area is not present in the man and hence the adverse effects were considered not significant to the man. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 175 mg/kg bw/day for animals of either sex.

Inhalation

Currently no study to assess toxicity of Basic Red 46 on repeated inhalation exposure is available. However, Basic Red 46 can be considered to have low volatility based on physical-chemical properties. The low partition coefficient, again points to poor absorption across the respiratory tract. Basic Red 46 was found to have high water solubility, hence the inhaled vapours may be retained within the mucus. The use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Further, Basic Red 46 has been investigated for adverse effects on repeated dose exposure via oral route in an OECD 422 study. Taking into consideration the above arguments, no elevated toxicity is expected via the inhalation route and safety for human health can be estimated using the route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Basic Red 46 is considered to be scientifically not necessary.

Dermal

Currently no study to assess toxicity of Basic Red 46 on repeated dermal exposure is available. However, Basic Red 46 is rather large in molecular size for dermal absorption. Further, owing to the high water solubility and the low partition coefficient, Basic Red 46 is considered too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, dermal uptake for the substance can be considered to be low. No systemic effects were observed in the acute dermal toxicity study and the skin irritation study with the substance. Further, Basic Red 46 has been investigated for adverse effects on repeated dose exposure via oral route in an OECD 422 study. Taking into consideration the above arguments, no elevated is expected via the dermal route and safety for human health can be estimated using the route to route extrapolation. Hence, the conduct of repeated dose toxicity study via dermal route for Basic Red 46 is considered to be scientifically not necessary.

Justification for classification or non-classification

The adverse effects observed in the repeated dose toxicity study with reproductive/developmental screening test were considered to be rodent-specific and not relevant to man, hence Basic Red 46 does not warrant classification according to the Regulation (EC) No. 1272/2008 (CLP).