Registration Dossier

Administrative data

Description of key information

Numerous repeated dose toxicity studies have been performed in rats, which include the following:  one 21-day repeated dermal toxicity study (Hixson 1981), two 28-day dietary studies (Naismith 1986; Bolus 1989), two 90-day dietary studies (Steinhoff and Gunselmann, 1986; Naismith 1987), one chronic gavage study (Steinhoff and Gunselmann, 1986), and two chronic dietary studies (Steinhoff and Gunselmann, 1986; Ciofalo 1992).  No systemic toxicity was observed up to the limit dose in the 21-day repeated dermal toxicity study (Hixson 1981).  The most consistent endpoint affected in the subchronic and chronic oral toxicity studies was decreased bodyweight.  The target organ of toxicity in subchronic and chronic studies was the pancreas.

Key value for chemical safety assessment

Additional information

The pancreas was identified as the target organ of toxicity in the repeated-dose studies that performed histopathological examinations. The changes in the pancreas generally consisted of multifocal acinar degeneration. These effects were identified as early as 14 days after commencement of treatment in a sub-acute study (Bolus 1989). Pancreatic toxicity was also observed in one of the sub-chronic and chronic studies, along with secondary signs of toxicity, including cataracts (Naismith 1987; Ciofalo 1992). Glucose deregulation was also identified in the chronic study (Ciofalo 1992). 

The following standard subchronic and chronic toxicity studies have been performed in rats:  one 21-day repeated dermal toxicity study (Hixson 1981), two 28-day dietary studies (Naismith 1986; Bolus 1989), two 90-day dietary studies (Steinhoff and Gunselmann, 1986; Naismith 1987), one chronic gavage study (Steinhoff and Gunselmann, 1986), and two chronic dietary studies (Steinhoff and Gunselmann, 1986; Ciofalo 1992). 

  

No systemic toxicity was observed in the 21-day dermal toxicity study at the limit dose of 100 mg/kg-day, although slight dermal irritation was observed with animals receiving 10 or 100 mg/kg-day (Hixon 1981). 

Naismith (1986) administered DETDA for 28 days in diet at concentrations of 40, 400, 800, 1200, or 3200 ppm. Extreme weight loss occurred in all animals receiving DETDA at 3200 ppm, which necessitated their early sacrifice on day 17. Excluding the 3200 ppm groups, the following daily intakes were calculated for male and female rats based on feed consumption: 40 ppm (males = 16 mg/kg bw/day; females = 19 mg/kg bw/day), 400 ppm (males = 42 mg/kg bw/day; females 43 mg/kg bw/day), 800 ppm (males = 80 mg/kg bw/day; females = 112 mg/kg bw/day), and 1200 ppm (males = 77 mg/kg bw/day; females = 124 mg/kg bw/day). Naismith (1986) identified the following effect levels: clinical signs (NOEL male = 42 mg/kg bw/day; female 43 mg/kg bw/day), decreased body weight (NOEL male = 16 mg/kg bw/day; female 19 mg/kg bw/day), increased food consumption (NOEL male = 42 mg/kg bw/day; female 43 mg/kg bw/day), increased relative liver-to-body weight (NOEL male = 80 mg/kg bw/day; females = 124 mg/kg bw/day), and gross pathology (NOAEL male = 77 mg/kg bw/day; females = 124 mg/kg bw/day). Histopathology was not performed. The most sensitive endpoint reported in this study was decreased bodyweight in male and female rats with NOELs of 16 mg/kg bw/day and 19 mg/kg bw/day, respectively.

Bolus (1989) performed a 28-day study with dietary concentrations of DETDA of 0, 50, 125, or 320 ppm. After 28 days, five animals per dose group were maintained on DETDA-free diet until day 56 to evaluate reversibility. Based on feed consumption, the following daily intakes were calculated for male and female rats: 50 ppm (males = 4 mg/kg bw/day; females = 5 mg/kg bw/day), 125 ppm (males = 10 mg/kg bw/day; females = 12 mg/kg bw/day), and 320 ppm (males = 24 mg/kg bw/day; females = 28 mg/kg bw/day). The following primary effect levels were reported: clinical signs (NOAEL males = 10 mg/kg bw/day; NOEL females = 28 mg/kg bw/day), decreased bodyweight (NOAEL male = 4 mg/kg bw/day; females = 5 mg/kg bw/day), and pancreatic multifocal acinar degeneration (NOAEL males = 4 mg/kg bw/day; females 5 mg/kg bw/day). The pancreas was identified as the target organ of toxicity. Pancreatic toxicity was observed in male and female rats in the mid- and high-dose groups at the 14-day, 28-day, and 56-day sacrifices, with progression of severity observed in three out of five male rats in the high-dose group at the end of the recovery period.    

Steinhoff and Gunselmann (1986) administered DETDA to rats for 90-days in feed at concentrations of 100, 300, or 900 ppm. The corresponding daily intake values for both male and female rats were: 100 ppm (~5 mg/kg bw/day), 300 ppm (~15 mg/kg bw/day), or 900 ppm (~45 mg/kg bw/day). The general endpoints affected were consistent with those reported by Naismith (1986) and Bolus (1989), that is, clinical signs of toxicity were evident and body weight decreases were observed in all high dose animals. No significant changes in these latter parameters were observed with animals of either sex. Males were more affected than females. Gross pathological evaluations in the high dose animals did not reveal any treatment-related effects. Since histopathology was not performed, the reproducibility of the pancreatic toxicity reported by Bolus (1989) could not be verified.

Naismith (1987) administered DETDA to rats for 90 days at concentrations of 50, 125, or 320 ppm in feed. The daily intakes, based on feed consumption, were: 50 ppm (males = 8 mg/kg bw/day; females = 10 mg/kg bw/day), 125 ppm (males = 21 mg/kg bw/day; females = 27 mg/kg bw/day), or 320 ppm (males 122 mg/kg bw/day; females = 125 mg/kg bw/day). The following general signs of toxicity were noted in male and/or female rats: clinical signs (NOAEL males = 21 mg/kg bw/day; females = 27 mg/kg bw/day), and decreased bodyweight (NOEL males = 8 mg/kg bw/day; females = 10 mg/kg bw/day). Naismith (1987) performed histopathological evaluations on all organs and confirmed the pancreatic toxicity reported by Bolus (1989). Naismith (1987) observed minimal to moderate multifocal degeneration of the acinar cells in rats receiving the low- and mid-concentration of DETDA in diet. Unlike animals receiving the high-concentration of DETDA in diet, the effects observed in the low- and mid-concentration groups did not result in an adverse impact on health. Male and female rats receiving the high concentration of DETDA in diet, however, presented with diffuse atrophy of the acinar cells and vacuolization of the islet cells of the pancreas. Secondary signs of toxicity from the pancreatic toxicity were observed in high-concentration male and female rats, with a high incidence of bilateral cataractous changes in the eyes. Increased pigmentation was also observed in the liver and spleen of animals receiving the high concentration of DETDA in diet. 

Collectively, the subchronic studies have shown that DETDA’s target organ of toxicity is the pancreas (Naismith 1986, 1987; Bolus 1989; Steinhoff and Gunselmann 1986).  The 90-day study performed by Naismith (1987) demonstrated that significant toxicity occurred only in male and female rats at dose levels of 122 mg/kg bw/day or 125 mg/kg bw/day, respectively. 

Three chronic studies have been performed on rats using the test material.  Steinhoff and Gunselmann (1986) administered the test material by gavage or by diet.  Ciofalo (1992) administered the test material by diet only. As with the subchronic studies, the most consistent general findings of toxicity reported across these studies were clinical signs of toxicity and decreased body weight (Steinhoff and Gunselmann, 1986; Ciofalo, 1992). The study performed by Ciofalo (1992) confirmed the pancreas as the target organ of toxicity.

In the chronic gavage study, Steinhoff and Gunselmann (1986) administered dose levels of 4 or 12 mg/kg bw/day. These dose levels were reduced at day 127 to 2 or 6 mg/kg bw/day due to significant clinical signs and reduced bodyweights. On day 256, the dose levels were reduced to 1 and 3 mg/kg bw/day because of continued signs of excessive toxicity. The bodyweights of female rats gradually returned to control levels; however, the bodyweights of male rats were reduced for the remainder of the study. A NOAEL of 3 mg/kg bw/day was identified for gross pathology and histopathology in male and female rats. No statistically or biologically significant changes in neoplastic lesions were observed between the treated and control animals of either sex.

In their chronic dietary study, Steinhoff and Gunselmann (1986) administered DETDA in diet at concentrations of 80 or 240 ppm. On day 465, the concentrations were halved to 40 and 120 ppm because it was apparent from the low body weights that the maximum tolerated dose of the test material was exceeded in animals originally receiving DETDA in diet at 240 ppm. The concentrations of 40 and 120 ppm corresponded to daily intakes of approximately 2 and 6 mg/kg bw/day. Though the body weights in the low concentration group improved, the body weights of male and female rats receiving DETDA at 120 ppm remained lower than the control animals until the end of the study. Despite this, no treatment-related or dose dependent pathological or non-neoplastic histopathological effects were observed (NOAEL = ~ 6 mg/kg bw/day). Though the total number of tumors in female rats was increased, no concentration-dependent response was observed and the increased values observed for tumors of the uterus and mammary gland were within those of the historical control data. In contrast, neoplastic lesions in the male rats were concentration-dependently lower than the corresponding observations in control animals. Based on the foregoing information, a study NOAEL ~2 mg/kg bw/day for body weight maintenance in male and female rats was observed, with NOAELs of 6 mg/kg bw/day being identified for nonneoplastic and neoplastic lesions.  

Ciofalo (1992) administered DETDA in diet to rats at concentrations of 10, 35, or 70 ppm for two years. The corresponding daily intake values for male and female rats were: 10 ppm (males = 0.4 mg/kg bw/day; females = 0.5 mg/kg bw/day), 35 ppm (males = 1.4 mg/kg bw/day; females = 1.8 mg/kg bw/day), or 70 ppm (males = 3.2 mg/kg bw/day; females = 3.8 mg/kg bw/day). Secondary clinical signs of pancreatic toxicity were observed in high dose males, with a few presenting with eye opacity. No additional treatment-related clinical signs were observed. Opthalmoscopic examinations revealed the presence of dense bilateral cataracts in 5 high dose male rats, which were first detected at 18 months. These observations correlated with 5 out of 6 male rats with nonfasting blood glucose values greater than 300 mg/dl. Further, 4 out of the 5 rats were subsequently diagnosed with multifocal pancreatic acinar cell atrophy, one with fatty infiltration of the pancreas, and one with multifocal interstitial fibrosis of the pancreas. These latter findings confirm the reported pancreatic toxicity from subchronic studies (Bolus 1989; Naismith 1987), and support that the pancreas is a target organ of toxicity following subchronic exposures and chronic exposures. 

Bodyweight was statistically significantly depressed in high dose male rats beginning on week 43 and continuing until study termination. An approximate 25% decrease in terminal bodyweight was recorded in high dose male rats compared to controls. Control and mid-dose bodyweights of male rats were statistically comparable throughout the study. The body weights of female rats were statistically comparable across groups, although terminal body weights of animals in the high dose group were decreased by ~13% compared to controls. Based on the body weight changes in high dose male and female rats, it may be concluded that the maximum tolerated dose of the test material was exceeded in male rats and nearly exceeded in female rats. 

On gross pathology, no treatment-related findings were observed. None to several macroscopic observations were made across groups, but were consistent with age-associated changes in this strain of rat. Histopathological findings in high dose animals included a significant increase in proliferative lesions in the liver and thyroid. In male rats, a statistically significant increase in hepatocellular carcinomas (18%; concordant control = 2%; historical control range = 0.77 to 6.67%) and thyroid follicular cell adenomas (10%; concordant control = 0%; historical control range = 1.67 to 12.00%) was observed in the high dose group. Though the percentage of high dose male rats presenting with hepatocellular carcinomas exceeded the laboratory’s historical control, several lines of evidence support that this finding was secondary to excessive toxicity from a high dose level.  First, the significant degree of general toxicity observed in this group of animals, as evidenced by the approximate 25% decrease in terminal body weights, indicates that the increased percentage of hepatocellular carcinomas was an indirect effect of treatment. Second, the percentage of animals presenting with hepatocellular carcinomas in the mid- and low- dose groups were well within the historical control values.  Finally, the studies conducted by Steinhoff and Gunselmann (1986) did not identify an increase in any particular tumor type; it is noteworthy that unlike Ciofalo (1986), these authors reduced the dose levels when excessive toxicity was observed. 

In high dose female rats, a statistically significant increase in hepatocellular adenomas (16%; concordant control = 4%; historical control range = 0.56 to 13.33%) was observed. In the high- and mid-dose females, a statistically significant increase in benign mammary gland tumors (fibroadenomas) was identified; however, the incidence of malignant mammary gland tumors (adenocarcinomas) was higher in the control females than in any of the treatment groups and no change in the time of onset of tumor formation was found for any group. Therefore, the relevance of the benign tumors is unclear. As with the liver tumors identified in the high dose male rats, the same lines of evidence support that the liver tumors found in high dose female rats were secondary to excessive toxicity from a high dose level.To start, female rats in the high dose group experienced a biologically significant decrease in body weight (~13%). Second, the percentage of animals with hepatocellular ademonas in the mid-dose female rats was within the historical control range, and below the percentage found in the concordant controls. Third, the two cancer studies performed by Steinhoff and Gunselmann (1986) did not identify treatment-related increases in tumors. Therefore, it was concluded that the increase in hepatocellular adenomas found in high dose females was secondary to generalized toxicity and not biologically relevant. 

In conclusion, the results of Ciofalo (1989) should be evaluated in conjunction with the studies performed by Bolus (1989) and Naismith (1986, 1987), as they were all performed at the same laboratory. The 90-day subchronic and 28-day progression/reversibility studies identified the pancreas as the target organ in the rat. Male rats were more severely affected, and at an earlier time and lower dose than female rats. Histologically, pancreatic acinar cells were affected first; islet cell involvement followed afterwards in a time and dose dependent manner. Pathology in other organs occurred after or in conjunction with islet cell involvement and appeared secondary to the metabolic changes induced by pancreatic toxicity. Secondary effects included ocular cataracts in animals with histological evidence of pancreatic toxicity and hyperglycemia. Histologic evidence of liver or thyroid toxicity was not observed in the sub-acute and sub-chronic studies. In the chronic study performed by Ciofalo (1992), histological evidence of pancreatic toxicity was restricted to the pancreatic acinar cells of high dose male rats. A functional aberration was suggested in individual rats by the hyperglycemia, increased food consumption, and ocular cataracts observed in some high dose male rats. No histologic or suggestive evidence of pancreatic toxicity was seen in the mid and low dose males or in the females at any dose level.  


Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: pancreas

Justification for classification or non-classification

Classification with STOT-RE according to CLP is comparable to the classification with R48/X. according to DSD. According to ANNEX I to DSD, containing the list of harmonised classification and labelling, which are legally binding, DETDA is classified R48/22. All substances with R48/22 shall be classified generally at least as STOT-RE Cat. 2. However, due to increase in guidance values and because the primary target organs for STOT-RE should be stated a re-evaluation is necessary.

Several studies are available for the assessment of the toxicity after repeated administration:

- 28 days studies (Naismith 1986; Bolus 1989)

- 90 days studies (Steinhoff and Gunselmann, 1986; Naismith 1987)

- Combined chronic /carcinogenicity studies (Steinhoff and Gunselmann, 1986; Ciofalo 1992)

The pancreas was identified as the target organ of toxicity in all repeated dose studies. Assessment:

For a 28 days study cut-of values of 300 and 30 mg/kg have been regarded for STOT-RE Category 1 versus Category 2 respectively. 

For a 90 days study cut-off values of 100 and 10 mg/kg have been regarded for STOT-RE Category 1 versus Category 2 respectively. At doses above 100 mg/kg, Naismith (1987) reported significant toxicity, whereas only mild to moderate evidence of toxicity was observed at doses within the STOT-RE Category 2 range. 

For a 12-month study, cut-off values of 25 and 2.5 mg/kg have been regarded for STOT-RE Category 1 versus Category 2 respectively. At the dose level of 3.2 (m) and 3.8 (f) significant/severe pancreatic toxicity was observed. The pancreas of the high dose males had a significant increase in multifocal acinar atrophy accompanied by interstitial fibrosis and fatty infiltration (replacement of glandular elements by adipose tissue). Secondary signs of toxicity, which included bilateral cataracts and elevated nonfasting blood glucose levels, accompanied these changes. Therefore, a classification in Category 2 is warranted. Though not explicitly stated in the criteria the “…study with the longest duration should normally be used”:

Classification:

Based on the evaluation of the 3-month and more relevant 2-year study, a classification in Category 2 without specifying a route of exposure is warranted.