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Description of key information

There are no subchronic or chronic repeated dose study data and reproduction toxicity data available for 7PPD. A read across approach with data from 6PPD is conducted.
7PPD and 6PPD have similarities in the chemical structure and physico-chemicals properties. These similarities include vapour pressures, low water solubility, a lipophilic character and hydrolytically instability. However, at room temperature the physical states are different. The similarities are also seen in mammalian cell toxicity. The skin and eye irritation potential for 7PPD and 6PPD is low. The in vitro and in vivo genotoxic potential is low. The acute oral toxicity for 7PPD is low and for 6PPD is moderate. For both substances a low dermal acute toxicity was indicated. Clinical signs and death occurred after oral and dermal application, indicating systemic availability for both application routes and substances. There is only a limited subacute feeding study available for 7PPD; whereas several repeated dose toxicity studies are available for 6PPD. For the 7PPD a NOAEL of 1500 ppm (males: 101.2 mg/kg bw, females: 134.3 mg/kg bw) was suggested, which based on slight effects on body weights and liver weights at 3000 ppm (males: 186.9 mg/kg bw/d, females: 199.6 mg/kg bw/d). For the 6PPD data from several repeated dose toxicity studies are available. Oral administration of 6PPD to male and female rats revealed adverse effects on the liver and changes in hematology. For the 6PPD a NOAEL of 20 mg/kg bw/day was oberseved within the subchronic and chronic studies.This was used as starting point for DNEL calculation .

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
chronic
Species:
rat

Additional information

The repeated dose toxicity of 7PPD was evaluated in a limited subacute feeding study with Sprague-Dawley rats (Monsanto 1988). Male and female rats (5 per sex and dose group) were dosed with 0, 500, 750, 1500 and 3000 ppm (ca. 0, 36.7, 51.8 101.2 and 186.9 mg/kg/day in males and 0, 39.8, 58.2, 134.3 and 199.6 mg/kg/day in females) in the diet for 28 days.

The stability and homogeneity of the test substance neat and when mixed with the diet were analysed. Overall averages of the dietary concentrations for the study were 450, 660, 1300 and 2800 ppm for the four treatment groups. Occurrence of clinical signs, body weight, and body weight gain and food consumption were recorded on a weekly basis; mortality and moribundity twice daily. At study termination gross pathology was performed and liver weights were determined for all animals. No histopathology, clinical chemistry and haematology were conducted. No mortality or clinical signs occurred during the study. Males and females at the highest dietary level had a decrease in group mean body weight (study mean: males -13.7 %, females -11.4 %). Additionally, reduced weight gains were observed in males and females at all but the lowest level males for the remainder of the study. Females at the lowest dietary level were slightly (about 10%) lighter in group mean body weight at the end of testing, while there were no significant differences in the lowest level males at any time during the study, when compared to controls. Food consumption was reduced for males at all but the lowest level and females at all dietary levels, except for 1500 ppm females on a gram per day basis. However these females failed to gain as much weight as the controls. No treatment-related gross lesions were noted in any of the treated animals. Slightly increased absolute liver weights occurred in males and females of the highest dose group (males + 6%, females + 10 %). Increased relative liver weights were seen in males and females at the highest dose group. Increases in relative liver weights were also seen in lower dose groups, but were attributed to decreases in body weights at those levels. The authors of the study suggested an NOEL of 500 ppm (males: ca. 36.7 mg/kg bw/d, females: ca. 39.8) which based on very slight body weight effects seen in the higher dose groups. The observed effects on the body weights are accompanied by a decreased food consumption of treated animals. Because no substance-related gross pathological lesions were noted in any of the treated animals, it is assumed that the decreased food intake is more an effect of non-palatable diet than of adverse effects on digestion system. However without histopathology, clinical chemistry and haematology the sensitivity of the study remains limited. A LOAEL of 3000 ppm (males: 186.9 mg/kg bw/d, females: 199.6 mg/kg bw/d) is suggested, which is based on body weight reduction and effects on liver weights observed in animals of the highest dose group; a NOAEL of 1500 ppm (males: 101.2 mg/kg bw/d, females: 134.3 mg/kg bw/d) is suggested.

There are no subchronic or chronic repeated dose study data and reproduction toxicity data available for 7PPD. A read across approach with data from 6PPD (N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine) is conducted (analogue approach see discussion below, data matrix of 7PPD and 6PPD in chapter toxicokinetics).

6PPD repeated dose toxicity data:

In in a GLP and guideline study (Guidelines for 28-Day Repeat Dose Toxicity Testing of Chemicals, Japan) in which 5 males and 5 female Sprague Dawley rats were exposed to 6PPD by gavage. The animals were treated with 0, 4, 20 and 100 mg/kg bw and day for 28 days. The control and 100 mg/kg bw and day group included additional 5 males and 5 females that were observed for further 14 days after termination of exposure. From the English summary and tables (detailed publication in Japanese) data on mortality, body weight, organ weights and microscopic and macroscopic are give. In addition, data of haematology, clinical chemistry and urinalysis are available. No effects on survival or body weight gain were reported. In rats doses with 100 mg/kg bw and day relative liver weights were significantly increased for both sexes at the end of administration accompanied by periportal fatty change. During recovery these effects were distinctly reversible but the increase of liver weight was still significant for male rats. The histopathological liver change was also observed in all females of the 20 mg/kg bw and day group, without an increase of liver weight. A significant increase of total serum protein was observed in females given 20 mg/kg bw and day or more (dose-dependent effect) and in males given 100 mg/kg bw and day at the end of administration. No such significant increase was noted in males and females of the highest dose group at the end of the recovery period. Increased proteinuria was found in the high dose group (both sexes), however, in the absence of histological changes of the kidney. There were no effects on weights or histopathologic findings of any other organs. In the high dose group further changes of clinical chemistry and haematological parameters were reported indicating an existing anaemia. The authors derived a NOEL of 4 mg/kg bw and day for this study (Hatano Research Institute 1999). As discussed in OECD SIDS (2005) a sex-specific sensitivity is obvious from the study data, the NOEL of 4 mg/kg bw and day is only valid for the female rats and the effects observed at the LOEL of 20 mg/kg bw and day were of a rather mild nature (reversible periportal fatty change of the liver without an increase of liver weight, increased total serum protein). In contrast, there were adverse effects on a range of different parameters observed at 100 mg/kg bw and day for both sexes so that the LOAEL for both sexes is rather at the dose of 100 mg/kg bw and day and the NOAEL at 20 mg/kg bw and day.

The toxicity of 6PPD was evaluated in a 13-week study with Sprague-Dawley rats (Monsanto Co. 1987). Males and females (25 per dose and sex) were feed with dosages of 0, 250 1000 or 2500 ppm 6PPD (males: 0, 15.7, 62.3, and 153.8 mg/kg bw/day; females: 0, 18.5, 75.0 and 172.1 mg/kg bw/day). The animals were examined for clinical signs, weight gain, food intake and mortality. Ophthalmic examinations, clinical chemistry and haematological examinations were done. At necropsy weights of brain, kidney, liver, spleen and testes were recorded and an extensive macroscopic and microscopic examination of all organs was performed. No mortality occurred and no test-substance related clinical signs were observed. Animals of the highest dose groups (1000 ppm, 2500 ppm) showed reduced body weight gains. This reduction in body weight gain was accompanied by reduced food consumption. At the 1000 ppm and 2500 ppm dose groups changes in clinical chemistry parameters (increase in total protein albumin, globulin, calcium and/or cholesterol for both sexes and increase in total bilirubin in males) were observed. Decreases of SGOT, SGPT, creatinine, and BUN values were also observed sporadically in either or both sexes, usually at the higher dietary levels. However, the biologically relevance of these decreases are questionable. Changes in haematology were seen in mid and high dose animals (anemia, lymphocytopenia and thrombocytosis). The authors concluded that the observed anemia was not caused by decreased red blood cell production but rather by an increased rate of red blood cell destruction due to the absence of histological lesions in bone marrow. Increased relative and/or absolute liver weights were noted at 1000 ppm and higher (25 % and 45 % increases in rel. liver weights at 1000 ppm and 2500 ppm, respectively). These increases were not accompanied by macroscopic or microscopic lesions. Decreased weights of testes and spleen (females) at the high dose in the absence of microscopic changes were not considered as treatment related. Female rats of the low dose group (250 ppm) had mild anemia at interim period sampling (study week 6 to 7) that was reversible within the end of study. Lymphocytopenia was observed in females from all dose groups at the terminal sampling. The toxicological significance and relationship to treatment of thrombocytois and lymphocytopenia in this study are unknown. From these data a NOAEL of 250 ppm (15.7 mg/kg bw/day males, 18.5 mg/kg bw/day females) was suggested, which based on anemia and increase of liver weights at the higher dose groups (LOAEL ≥ 62 mg/kg bw day).

In a chronic two-year study rats were feed with 0, 100, 300 or 1000 ppm 6PPD in the diet (ca. 0, 8, 23 and 75 mg/kg bw/day) (Monsanto Co.1978). Animals were observed daily for mortality and toxic signs. Body weights were measured weekly for 13 weeks and monthly thereafter. Food consumption was measured from dietary level weekly for the first 13 weeks and for one week in each month of the study thereafter. Ten animals per sex from control and high dose groups were examined at 3, 6, 12, and 18 months for changes in haematology, clinical chemistry and urinalyses; ten animals per sex from each group were also examined for changes in these parameters at 12 and 24 months. Complete gross necropsies were conducted on animals found dead, on all animals sacrificed in extremis and on all animals surviving at 24 months. Organ weights and organ/body weight or organ/brain weight were recorded for brain, gonads, heart, kidneys, liver, and spleen from all animals sacrificed at 24 months. Histopathological examinations were conducted on all common organs. Survival of treated animals was comparable to control animals. No test substance-related clinical signs were observed. Body weights and body weight gains were reduced for males and females of the highest dose group. For females, this persisted throughout most of the study and was statistically significant (p<0.01) for the first 16 months of the study. Body weights and body weight gains for mid and low dose groups were comparable to control groups. Food consumption was lower during the first two weeks for females of the high dose group and to the first four weeks of the study for males of the highest dose group. No test substance-related changes were noted in clinical chemistry and urinalyses. At some interim intervals changes in haematological parameters were revealed. Erythrocyte counts were lower for high dose males at 3 months and high dose female at 3, 6, and 12 months. Hemoglobin concentration was statistically reduced for high dose males at 3, 12 and 18 months and for high dose females at 6, 12 and 18 months. Although reduced, most haemoglobin concentration results were considered within the normal range. Hemocrite values were significantly lower than control for high dose females at 3, 6 and 12 months. However, a repeat of the hematologic analysis at 12 months using all animals from test groups revealed no statistically significant differences compared to control. The hemocrite values increased slightly for the high dose females at 18 and 24 months. In high dose males an increase in liver weights (19 %) were seen and in high dose females a decrease in kidney (11 %) and spleen weights (22 %) were noted. A final examination gave no indication for gross pathological findings, histopathological changes and neoplastic alterations caused by 6PPD. Based on the findings obtained (reduced body weight and changes in haematology) a NOAEL of 300 ppm (ca. 23 mg/kg bw/day) is suggested.

In a second chronic study (Monsanto 1993b) 6PPD was administered orally, via dietary admixture to Spraque –Dawleys rats at dose levels of 0, 50, 250, and 1500 ppm. Homogeneity, stability and actual dietary concentrations were analytically verified throughout the study and confirmed to be within the range of ± 15% of the nominal concentration. The mean test-substance intake over the duration of the study was as follows:

- 50 ppm: 2.6 mg/kg/day (males) and 3.2 mg/kg/day (females)

- 250 ppm: 13.5 mg/kg/day (males) and 16.5 mg/kg/day (females)

- 1500 ppm: 84.8 mg/kg/day (males) and 109.5 mg/kg/day (females)

At the beginning of the experiment 70 rats/sex/group were dosed with 6PPD. At 12 months of treatment approximately 20 rats/sex/group were sacrificed. After 24 months of treatment all survivors were sacrificed. Ophthalmoscopic examinations were reported after 12 months and at study termination. Body weight and food consumption were recorded weekly throughout the first 13 weeks and monthly thereafter. Hematology (20 animals per time point/sex/group), clinical chemistry (10 animals per time point/sex/group), and urine analysis (10 animals per time point/sex/group) were performed prior to study initiation, during months 3, 6, 12, 18, and at termination. Blood was taken after fasting over night, urine was taken from unfastened, water depreciated animals and the following parameters were reported. Hematology: hemoglobin concentration, hematocrit, erythrocyte counts, platelet counts, mean corpuscular volume, mean corpuscular hemoglogin, mean corpuscular hemoglobin concentration, total and differential leucocyte counts. Clinical chemistry: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, blood urea nitrogen, fasting glucose, cholesterol, total protein, albumin, globulin, albumin to globulin ratio, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, creatinine phosphokinase, gamma glutamyl transpeptidase. Urine analysis: specific gravity, protein, pH, glucose, ketones, bilirubin, occult blood, urobilinogen, 16h volume, microscopic analysis. Post mortem gross examination was reported on all animals. Organ weights were recorded for ≥ 10 animals/sacrifice timepoint/sex/dose. The following organs were weighted: adrenals, brain, kidneys, testes with epididymides, liver, ovaries, and spleen. Histopathologic evaluation of all animals in the control and high dose groups were performed and on selected organs in the interim groups. The following organs were evaluated in the control and high-dose groups: adrenals, aorta (abdominal), bone (including the articular surface, sternum and femur), bone marrow (sternum), brain (medulla/pons, cerebellar cortex and cerebral cortex), esophagus, eyes, heart, intestine, cecum, colon, duodenum, ileum, jejunum, rectum, kidneys, liver, lungs (including trachea) – inflated with fixative, lymph nodes (mesenteric, mediastinal), mammary gland (right inguinal), ovaries, pancreas, peripheral nerve – sciatic, taken with biceps femoris, pituitary, accessory genital organs (prostate, seminal vesicles, epididymides), salivary glands (submandibular), skeletal muscle (right biceps femoris), skin, spinal cord (midthoracic, lumbar, cervical), spleen, stomach, testes, thymus, thyroid (including the parathyroids), urinary bladder – inflated with fixative, uterus, all gross lesions and tumors.

There were no statistically significant differences among survivorship or early deaths in any dose group compared to controls. Clinical observations were of the type commonly seen in lifetime rodent studies and no consistent patterns were evident. There was an increase in the incidence of poor condition and emaciation of the high dose females, but not males. No treatment related ophthalmoscopy observations were reported after 12 months or at termination. Mean body weight was consistently lower in males and females at 1500 ppm (mean difference to control – 9.9% and -18.4%, respectively) and lower in females at 250 ppm (-5.4%). Mean food consumption was increased in males and females at 1500 ppm (mean difference to control +5.5% and +17.3%, respectively) and in females at 250 ppm (+4.1%).

Hematologic observations indicate that 6PPD might induce a slight anemia in animals of the high dose groups. Body and liver weights are already affected at the mid dose groups. No histopathologic observations are reported for any blood forming organ; absolute and relative spleen weights are increased only in the high-dosed males at 12 months, but not at termination. Taken together the hematologic observations are considered slight, high-dose effects with no morphologic correlates.

Consistent statistically significant increased cholesterol levels were observed in males and females in the high dose groups at 6, 12, 18, and 24 months.

There were no consistent observations in in urine analysis for any parameter measured.

Mean absolute and relative kidney weights were increased in the high-dose groups in males and females at 12 months, but not at termination. Chronic nephropathy correlated with and increased incidence of irregularities of the kidney surface observed macroscopically in males and females at the high dose groups. The incidence of chronic nephropathy was similar between the control groups and the dose groups. However, the severity of chronic nephropathy increased among the high-dosed animals of both sexes sacrificed after 12 months and at termination. Mean absolute and relative liver weights were increased in males and females after 12 and 24 months in the high-dose groups and in the mid-dose groups at termination. Microscopic observations indicate increased incidence in pigment in the hepathocytes and reticuloendothelial cells and cytoplasmic vacuolization of the liver in females at the high dose. No effect was observed in males or females in the mid dose or males in the high dose. Follicular cell hyperplasia in the thyroid was slightly but not statistically significant increased among treated males. For further details on this observation see chapter carcinogenicity.

Overall, the No-observed-effect-level (NOEL) of this oral chronic toxicity study is 50 ppm in males and females (2.6 mg/kg/day in males, 3.2 mg/kg/day in females), based on reduced body weight in females, increased food consumption in females and increased liver weight at 250 ppm in both sexes (13.5-16.5 mg/kg/day). The observations in the mid dose are not regarded to be clearly adverse. At the high dose (1500 ppm; 84.8-109.5 mg/kg/day) adverse liver histopathology in females and slight effects on hematology are observed in both sexes.

In conclusion:

The repeated dose toxicity of 7PPD was evaluated in a limited subacute feeding study with Sprague-Dawley rats (Monsanto 1988) (as discussed above). Based on findings of this study a NOAEL of 1500 ppm (males: 101.2 mg/kg bw, females: 134.3 mg/kg bw) is suggested, which based on effects on body and liver weights at the highest dose group (3000 ppm) evaluated.

There are no subchronic or chronic repeated dose study data and reproduction toxicity data available for 7PPD. A read across approach was conducted with 6PPD (N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine) (see discussion below, data matrix analogue approach see chapter toxicokinetic).

Read across approach

7PPD and 6PPD have a common chemical structure, which consists of N-phenyl-para-phenylenediamine with different side chains at the second nitrogen (6PPD: 1,3-dimethylbutyl groups, 7PPD: 1,4-dimethylpentyl groups).

7PPD is a solid with a melting point of 29.8°C to 34.9°C; whereas 6PPD has a solid state with a melting point of 49.2°C. Both substances have very low vapour pressures (7PPD 0.00000281 hPa at 25°C Currenta, 2009; 6PPD 0.0000066 hPa at 25°C). The water solubility of both substances is low (7PPD: 0.67 mg/l at 25°C; 6PPD: 1.1 mg/kg/l at RT. No Log Pow could be experimental determined because both substances are hydrolytically unstable at pH 7, with a half-life of 5.15 h and 8 h for 7PPD and 6PPD, respectively. However, the calculated Log Pow is 5.71 for 7PPD and 4.68 for 6PPD. The major hydrolysis product for both substances is the 4-hydroxydiphenylamine.

In conclusion, 7PPD and 6PPD have similarities in the chemical structure. These similarities are seen in comparable physico-chemical properties like very low vapour pressures, low water solubility, a lipophilic character and hydrolytically instability. However, at room temperature the physical states are different.

The similarities seen in the physico-chemical properties of both substances are also reflected in mammalian toxicity of 7PPD and 6PPD. Both substances showed a very low skin irritating- and an even mild eye irritating potential.

The in vitro mutagenic potential of both substances is very low, whereas weak clastogenic effects in vitro were observed; no in vivo genotoxic potential was indicated neither for 7PPD nor 6PPD.

The acute oral toxicity of 7PPD is low indicated by an oral LD50 of 2100 mg/kg bw. Treated rats exhibited clinical signs like reduced appetite and activity. Mortality occurred in all treatment groups (Monsanto Co. 1973). In an earlier acute toxicity study with 7PPD an oral LD50 of 2170 mg/kg bw was calculated (Monsanto Co. 1967a).

The acute oral toxicity of 6PPD was evaluated in a GLP and OECD guideline study (TG 401) (Hatano Research Institute 1999, Ohara 1999). 6PPD suspended in corn oil was orally administered by gavage. Treated animals showed clinical signs like decreased fecal output, fecal/urine stains, rough coat, piloerection, diarrhea/soft stools and dark material around the facial area. Death occurred in the highest dose groups evaluated (1000 mg/kg bw and 2000 mg/kg bw). An oral LD50 value of 1005 mg/kg bw for males and 893 mg/kg bw for females were calculated. In another GLP and guideline study Sprague-Dawley rats were orally dosed with warmed 6PPD (Monsanto 1991). Based on findings from this study an acute oral LD50 greater than 5000 mg/kg bw was estimated. Earlier studies from 1962 and 1973 reported oral LD50 values in the range of 3340 - 3580 mg/kg bw (Monsanto Co. 1962, 1973). The inconsistent range of oral LD50 values for the 6PPD can be explained by the substance preparation which influences the bioavailability after oral application.

The dermal acute toxicity is low for both substances. For the 7PPD a dermal LD50 value in rabbits of >5010 mg/kg was calculated for 7PPD (Monsanto Co. 1973). Treated animals exhibited clinical signs and mortality occurred in the highest dose group. In an earlier study a dermal LD50 > 10000 mg/kg was estimated. For the 6PPD a dermal LD50 >7940 mg/kg bw was calculated (Monsanto Co. 1973). Rabbits treated with 6PPD showed clinical signs like reduced appetite and activity. No mortality occurred after dermal application of 6PPD (Monsanto Co. 1973). In an earlier study with rabbits mortality occurred at doses > 5010 mg/kg. Clinical signs like loss of appetite, lethargy and gradual wasting were observed (Monsanto Co. 1962).

The repeated dose toxicity of 7PPD was evaluated in a limited subacute feeding study with Sprague-Dawley rats (Monsanto 1988) (as discussed above). Based on findings of this study a NOAEL of 1500 ppm (males: 101.2 mg/kg bw, females: 134.3 mg/kg bw) is suggested, which based on effects on body and liver weights at the highest dose group (3000 ppm) evaluated.

Several oral repeated dose toxicity studies were conducted to evaluate the toxicity of 6PPD (as discussed above). In a subacute gavage study (Hatano Research Institute 1999) effects on liver weights accompanied by histological changes were noted in treated rats. In high dose group animals changes in clinical and haematological parameters were reported, which indicate an existing anemia. Based on the reversibility and severity of liver changes a NOAEL of 20 mg/kg bw and day was suggested. In a subchronic feeding study with rats a NOAEL of 250 ppm (15.7 mg/kg bw/day males, 18.5 mg/kg bw/day females) was suggested, which based on anemia and increased liver weights at higher dose groups (Monsanto 1987). In a chronic feeding study with rats (Monsanto 1978) a NOAEL of 300 ppm (ca. 23 mg/kg bw/day) was suggested, which based on reduced body weight and body weight gain, changes in haematology and organ weights at higher dose groups. In a second chronic study (Monsanto 1993b) liver histopathology and effects on hematology were reported at 1500 ppm (84.8 - 109.9 mg/kg/day).

In summary, oral administration of 6PPD to male and female rats revealed adverse effects on the liver and changes in hematology. Based on the findings from the repeated dose toxicity studies a NOAEL of 20 mg/kg bw/day is taken.

Justification for classification or non-classification

Based on the criteria defined in the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012. Classification as STOT RE is not appropriate because the reported effects in repeated dose toxicity studies are reversible and/or “not significant or severe” and/or at dose level above the cut-off values.

7PPD subacute toxicity study:

According to the CLP criteria effects below a cut-off value of 300 mg/kg/day should be further evaluated for subacute toxicity studies.

In the 7PPD study a NOAEL of 1500 ppm (males: 101.2 mg/kg bw, females: 134.3 mg/kg bw) is derived and slight effects on body weights and liver weights were observed at 3000 ppm (males: 186.9 mg/kg bw/d, females: 199.6 mg/kg bw/d), a dose wery close to the cut-off dose mentioned above. As outlined in theECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012, Annex1: 3.9.2.8.1 small changes in body weight and changes in organ weight without evidence of organ dysfunction do not support classification for STOT RE, although it is recognized that the study is limited because no histopathology is performed.

6PPD subacute toxicity study:

According to the CLP criteria effects below the cut-off value of 300 mg/kg/day should be further evaluated for subacute toxicity studies.

Rats were treated with 0, 4, 20 and 100 mg/kg/day. The No-Observed-Effect Level was reported to be 4 mg/kg/day. Based on the above mentioned threshold effects at 20 and 100 mg/kg/day should be further evaluated. The study includes a recovery group which additional 5 males and 5 females that were observed for further 14 days after termination of exposure to the high dose of 100 mg/kg/day. Liver weights were increased and periportal fatty changes were reported. During recovery these effects were reversible. A significant increase of total serum protein was observed in females given 20 mg/kg bw and day or more (dose-dependent effect) and in males given 100 mg/kg bw and day at the end of administration. No such significant increase was noted in males and females of the highest dose group at the end of the recovery period. Slightly increased proteinuria was found in the high dose group (both sexes), however, in the absence of histological changes of the kidney. As outlined in the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012, Annex1 3.9.2.5.5 recovery groups should be taken into account to judge reversibility; reversible observations should not lead to classification. Since all effects observed in this study do not justify classifation based on the criteria defined in the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012, Annex 1: 3.9.2.8.1. and/or are reversible according to the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012, Annex 1: 3.9.2.5.5 classification for STOT RE is not supported by this comprehensive sub acute toxicity study including a recovery group.

6PPD subchronic toxicity study:

According to the CLP criteria effects below a cut-off value of 100 mg/kg/day should be further evaluated for subchronic toxicity studies.

In this study rats were feed with dosages of 0, 250 1000 or 2500 ppm 6PPD (males: 0, 15.7, 62.3, and 153.8 mg/kg bw/day; females: 0, 18.5, 75.0 and 172.1 mg/kg bw/day). A NOAEL of 250 ppm (15.7 mg/kg bw/day males, 18.5 mg/kg bw/day females) was suggested. Effects at the mid-dose (62 mg/kg/day) will be further discussed.

Thrombocytois and lymphocytopenia was observed in females from all dose groups at the terminal sampling. The toxicological significance of this dose independent observation is unknown. At the 1000 ppm and 2500 ppm dose groups changes in haematology and clinical chemistry parameters were observed. However, the biologically relevance of these decreases are questionable at 1000 ppm because no kidney histopatholy was observed at any dose (1000 and 2500). Since the observations were dose dependent only slight effects are observed at 1000 ppm. Dose dependent increase in relative and/or absolute liver weights were noted at 1000 ppm and higher. These increases were not accompanied by macroscopic or microscopic lesions. Since small changes in clinical biochemistry and heamatology do not justify classification according to the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012,Annex1: 3.9.2.8.1 and liver weight increase without evidence of organ dysfunction or histopathology dose also not trigger STOT RE according to the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012,Annex 1: 3.9.2.8,classification is not supported by this comprehensive subchronic toxicity study.

6PPD chronic toxicity studies:

According to the CLP criteria effects below a cut-off value of 12.5 - 25 mg/kg/day should be further evaluated for chronic toxicity studies. According to the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012,Annex 1: 3.9.2.9.5 Haber’s rule should be applied for time extrapolation. According to the Example given in chapter 3.9.6.1.1, a cut-off value can be defined for a 1-year study by dividing 100 mg/kg/day with a factor of 4 (=25 mg/kg/day); consequently, factor of 8 might be applied for chronic 2 years studies in rats (100/8 = 12.5 mg/kg/day).

 

In an early chronic two-year study rats were feed with 0, 100, 300 or 1000 ppm 6PPD in the diet (ca. 0, 8, 23 and 75 mg/kg bw/day) (Monsanto Co.1978). Based on the findings obtained at 75 mg/kg/day (reduced body weight and changes in haematology) a NOAEL of 300 ppm (ca. 23 mg/kg bw/day) is suggested. Since the NOAEL, and consequently also the LOAEL, is above the cut-off value for chronc studies,classification is not supported by this early, limited chronic toxicity study.

 

In a second chronic study (Monsanto 1993b) 6PPD was administered orally, via dietary admixture to Spraque –Dawleys rats at dose levels of 0, 50, 250, and 1500 ppm (ca. 3, 14 and 85 mg/kg/day). Overall, the No-observed-effect-level (NOEL) of this oral chronic toxicity study is 50 ppm in males and females (2.6 mg/kg/day in males, 3.2 mg/kg/day in females), based on reduced body weight in females, increased food consumption in females and increased liver weight at 250 ppm in both sexes (13.5-16.5 mg/kg/day). The observations in the mid dose are not regarded to be clearly adverse. At the high dose (1500 ppm; 84.8-109.5 mg/kg/day) adverse liver histopathology in females and slight effects on hematology are observed in both sexes.As outlined in theECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012, Annex1: 3.9.2.8.1 small changes in body weight and changes in organ weight without evidence of organ dysfunction do not support classification for STOT RE.

Overall: Based on the cut-off values criteria defined in the ECHA Guidance on the Application of the CLP Criteria, Version 2.0, dated April 2012, classification is not appropriate because the reported effects in repeated dose toxicity studies are reversible and/or “not significant or severe” and/or at dose level above the cut-off value for classification.