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Key value for chemical safety assessment

Effects on fertility

Additional information

In a 2- year chronic feeding study with Sprague-Dawley rats of both sexes (Scientific Associates Inc. 1966; Val. 2), the animals were treated with 0, 11, 25 and 50 mg/kg bw/day of the test substance; it has to be noticed that the highest dose level of 50 mg/kg bw/day was increased step by step in the course of the study until reaching ca. 400 mg/kg bw/day.

At necropsy, deviations in organ weights were within the expected weight limits for the respective organs and no dose- response relationship could be evidenced. Gross pathology at 6 months was inconspicuous. At 12 months one case of slight testicular atrophy was grossly observed in one 200 ppm-dosed male. At 24 months, i.e. at the end of the study period, testicular atrophy was observed in 3/18 (17%) animals of the 200 ppm dose group, 2/17 (12%) animals of the 500 ppm dose group and 4/15 (27%) animals of the 1000-10,000 ppm dose group; no testicular atrophy was observed in the 34 control males which had been sacrificed at test ending. Only one high dose female had gonad atrophy, but the finding is considered to be fortuitous.

In the high dose group, 3 cases of gonad atrophy were grossly described as slight atrophy in one testis, and this was confirmed histologically; the fourth case was not grossly observed, but the histologic description indicated that 2/3 of the testis had a "moderately severe" generalized degree of atrophy present with only Sertoli cells which remained within most of the seminiferous tubules. According to the sponsor, the lack of testicular atrophy in the controls was surprising given the historical control range of this lesion (data not available). It should be noted that this is not an unusual lesion in ageing rats (Glaister, Principles of Toxicological Pathology, Taylor & Francis, London, 223 pp, 1986). It should be emphasized that in the six month samples, the more appropriate time point for examining reproductive toxicity, no testicular effects were observed. The atrophy often occurred bilaterally.

Despite the fact, that in the males of the high dose group, the incidence of testicular atrophy was clearly increased compared to control (27% versus 0%), the finding is considered to rather be related to the age of the animals than to be treatment-related. In fact, according to James RW and Heywood R (Age-related variations in the testes of Sprague-Dawley rats. Toxicol. Letters Vol 4(4): 257-261, 1979) spontaneous variations in testicular weight and histology are rarely encountered in rats maintained for 13- or 26-week observation periods, however, rats maintained for longer periods tend to show an increasing incidence of testicular atrophy. According to these 2 authors, changes in pituitary gonadotrophin secretion and androgen production observed in ageing male rats support the hypothesis that testicular atrophy is a senescent phenomenon. Thus, even if atrophy of rat testis is considered to be a useful index of chemical toxicity (Ribelin WE, Atrophy of rat testis as index of chemical toxicity, Arch Pathol 75: 229-235, 1963), age-related atrophy must be considered in studies exceeding 12 months’ duration as it is the case in the present study.

Thus, in accordance with the opinion reported in the OECD SIDS on DEAE (2004), the finding “testicular atrophy” was considered to be not treatment-related for the following reasons:

1) the finding was not dose-dependent (17% in the low dose, 12% in the mid dose and 27% in the high dose group);

2) a similar finding was not seen in the 6 month old males sacrificed at interim and at a time point considered to be the more appropriate to assess induction of gonad lesions (James and Heywood, 1979);

3) six months later, only one 12 month old male showed testicular atrophy, which further belonged to the mid dose group;

4) the finding is not an unusual one in aging rats (James and Heywood, 1979; Glaister, 1986);

5) referring to the absence of testicular atrophy in the control group (0%), the sponsor mentioned that the lack of testicular atrophy in the controls was surprising given the historical control range of this lesion; unfortunately, no historical control data were included in the study report. However, according to the OECD SIDS (2004), in a study conducted nearly 35 years later, it was reported that the incidence of testicular atrophy found in control 31 week old Charles River CrJ: (SD) rats was 20% (Sugimoto et al., Background data on organ weights and histopathological lesions in the Crj:CD(SD)IGS rats for 4-, 13-, and 26 week repeated-dose toxicity studies, in Maeda, Y. and Inoue, H. (eds.) Biological Reference Data on CD(SD) IGS Study Group - 2000, Yokohama, 2001: 79 - 87; also cited in OECD SIDS 2004). According to, the type of rat used in the 1960s was probably a Sprague-Dawley (personal communication, P.A. Mirley, 24 June 2002, cited in OECD SIDS 2004). Thus, the testicular finding in this study with 2 year old albino rats is not surprising, and the lack of testicular atrophy in the 34 control males most likely was a result of the randomness of the group assignments.

Thus, in present study, the findings referring to the reproduction organs are considered to be related to naturally-occurring disease processes common to aging laboratory rats and to be not treatment-related.

 

In a 1 year feeding study, groups of beagle dogs (3 males/3 females) were fed DEAE levels of 20, 40, 200, and 400 mg/kg bw/d for 365 days (Scientific Associates Inc. 1966; Val. 2). All animals at the high concentration died between 18 and 35 days of treatment. Animals at the second highest concentration were taken off treatment after 39 days. The 4 survivors from this group were administered 80 mg/kg bw/day of the chemical via gelatin capsules on days 134 through day 365. A control group received basal dog meal only. At necropsy, tissues examination of the animals treated with 80 mg/kg bw/day showed atrophy of the gonads for 3 males. Referring to the testes atrophy, Hottendorf and Hirth (1974) had examined the pathological findings of 1000 animals aged between 8 and 20 months for the purpose of establishing a pathologic profile of gross and microscopic spontaneous occurring lesions in Beagle Dogs (Hottendorf and Hirth, Lesions of spontaneous subclinical diseases in Beagle Dogs, Vet pathol Vol 11: 240-257, 1974). Especially referring to the reproduction organs, atrophy, hyperplasia, giant cell formation and epididymitis in the testes and/or prostate were reported as typical spontaneous occurring lesions. The authors pointed out that the findings indicate that the term “normal”, when applied to experimental Beagle dogs, is an extremely relative term, since the dogs considered for establishment of the pathological profile were young, commercially bred, and clinically healthy. Thus, according to the authors, attention must be given to the fact that spontaneous occurring alterations such as those described above may significantly affect a dog's reaction to high doses of drugs/toxicants and other stressful experimental procedures and must be invoked in the evaluation of experimentally induced changes.

James and Heywood also investigated age-related variations in the testes and prostate of Beagle Dog (James RW and Heywood R, Age-related variations in the testes and prostate of Beagle Dog, Toxicology Vol. 12: 273-279, 1979). Sufficient data were available to assess the age-related variations occurring in the testes and prostate of a sample of 198 male Beagles aged from 37 weeks to 7.75 years at the time of examination. Age-related differences were apparent in the distribution of total testicular weight. The results of this survey confirmed that spontaneous variations in the weight and morphology of the Beagle testes and prostate may influence the assessment of toxicological data.

In the present study, testes atrophy had been reported ported for the 3 male dogs initially treated with 5000 and then with 2000 ppm. Taking into consideration the observations reported above (Hottendorf and Hirth, 1974; James and Heywood, 1979) and the small number of animals used in the present study, and since severe toxicity was evidenced at the dose level where testes atrophy was seen, the finding was seen as non-specific secondary response to the metabolic or toxic insult of the test substance.

 

In a 14 week inhalation study according to U.S. EPA guidelines, 20 rats/dose/sex were exposed to 0, 53, 120 or 365 mg/m3 of DEAE for 6h/day, 5 days/week for 14 weeks using a whole body exposure method (Exxon 1990; Val. 1). Half of the animals were terminated at the end of week 14; the remaining animals were given a four week post-exposure recovery period prior to sacrifice.

With respect to the reproductive organs, histopathological examination of 10 animals /sex of the control and the high dose group, respectively, revealed following findings:

TEST ENDING (14 WEEKS):

Males:

- In the control group: 1/10 male with slight unilateral focal atrophy of the testes

- In the high dose group: 1/10 male with slight unilateral focal atrophy of the testes

Females:

- In the control group: 3/10 females with distended uterus; ovaries were inconspicuous.

- In the high dose group: 2/10 females with distended uterus; ovaries were inconspicuous.

 

AFTER RECOVERY:

Males:

- In the control group: 1/10 male with changes in the testes (no atrophy).

- In the high dose group: 1/10 male with slight focal periarteritis in the testes and 1/10 male with minimal atrophy of the testes.

Females:

- In the control group: 6/10 females with distended uterus; ovaries were inconspicuous.

- In the high dose group: 3/10 females with distended uterus; ovaries were inconspicuous.

Thus, in the present 14-week inhalation study with recovery, the findings in reproduction organs reported for the high dose treated animals of both sexes were almost similar to those reported for the untreated control animals.


Short description of key information:
No reproduction toxicity studies are available for DEAE. However, suitable data on reproductive organs are available from a 2-year repeated dose feeding study with rat (Scientific Associates Inc. 1967, Val 2.), a one-year repeated doses feeding study with Beagle dogs (Scientific Associates Inc. 1966; Val. 2), and a 14 weeks repeated inhalation study with rats (Exxon 1990; Val 1.).

Effects on developmental toxicity

Description of key information

Two prenatal developmental toxicity studies in rats conducted similarly to the OECD guideline 414 as well as a reliable range-finding study and one prenatal developmental toxicity studies in rabbits conducted in compliance with OECD 414 indicate that the test substance is not a developmental toxicant.

Effect on developmental toxicity: via inhalation route
Dose descriptor:
NOAEC
486 mg/m³
Additional information

Developmental toxicity:

In a developmental toxicity study performed according to OECD guideline 414 (under GLP), the test-substance was evaluated for its prenatal developmental toxicity in New Zealand White rabbits (2016). The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 15, 50 and 150 mg/kg on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (0.5% Carboxymethylcellulose suspension in drinking water (0.5% CMC)) in parallel. At terminal sacrifice on GD 29, 21-24 females per group had implantation sites. Maternal toxicity was evident in the high-dose group. In this dose groups, reduced food consumption (average 25% below control during treatment), reduced body weight gain (average 37% below control during treatment), lower carcass weights, increased aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities, increased triglyceride and inorganic phosphate levels and increased absolute (+22%) and relative (+26%) liver weights were observed. No test substance-related adverse effects in dams of the mid- and high-dose group was observed. In no dose group, test substance-related adverse effects in the fetuses were observed. Under the conditions of this prenatal developmental toxicity study, the NOAEL for maternal toxicity is 50 mg/kg bw/d, based on reduced food consumption, reduced body weight gain and liver weight increase together with changes in clinical chemistry at a dose of 150 mg/kg bw/d and the NOAEL for prenatal developmental toxicity is 150 mg/kg bw/d, the highest dose tested. Under the conditions of this study the test substance is not teratogenic in rabbits at the tested dose levels.

In prenatal developmental toxicity study similar to OECD guideline 414, timed-pregnant Sprague-Dawley rats were exposed whole body to DEAE vapor for 6 h per day on gestational days (GD) 6-15 at targeted concentrations of 33, 66 or 100 ppm (Leung 1998; Val. 1). Dams were sacrificed on GD 21. There was no maternal mortality in any exposed groups. Maternal toxicity observed in the 100 ppm group included dry rales, reduced body weight (9.5%) on GD 15 and reduced weight gain (48%) during exposure. The developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed. A statistically significant decrease in mean resorptions was observed in the100 ppm group. This finding was considered spurious and unrelated to DEAA treatment. No increases in malformations were observed. Therefore, the no-observed-adverse-effect level was set at 33 ppm for maternal toxicity (0.16 mg/L air) and at 100 ppm for embryo/fetal toxicity and teratogenicity (0.486 mg/L air).

In a range finding test to determine the dose-range for the above developmental toxicity study, groups of 8 pregnant rats were exposed to vapors of DEAE at concentrations of 0, 10, 50, 100, 150 and 200 ppm on gestation days 6 to 15 and were sacrificed on GD 21 (Leung 1998; Val. 2). The concentrations levels corresponded to ca. 0.05, 0.24, 0.48, 0.73, 0.97 mg/L air, respectively. Maternal toxicity was limited to rales and nasal or ocular discharge (150 and 200 ppm). Lost body weight and body weight gain was observed at 50 to 200 ppm. Pregnancy rates, mean uterine implantation data and fetal body weights were similar in all groups. External fetal malformations were limited to abnormal flexure of the hind-limb in one fetus each of the 50 and 100 ppm groups.Thus, the NOAEC for maternal toxicity was 100 ppm corresponding to 0.48 mg/L air; the NOAEC for developmental toxicity /teratogenicity was set at 200 ppm (0.97 mg/L).

 

In a second prenatal developmental toxicity study similar to OECD guideline 414, the test article was administered in deionized water by gastric intubation to four groups of five bred female rats once daily from gestation days 0 through 11 (Wil Research Laboratories Inc. 1997; Val.2). Dosage levels were 0, 10, 30, 100 and 250 mg/kg/day, respectively. All animals survived to the scheduled necropsy on gestation day 12. The only treatment-related clinical finding was rales in the 250 mg/kg/day group. No internal findings were observed at the macroscopic examination. Postimplantation loss was increased and the number of viable embryos was decreased in the 250 mg/kg/day group. Intrauterine parameters were unaffected by treatment in the 10, 30 and 100 mg/kg/day groups. Based on the results of this study, the no observed effect level (NOAEL) was 100 mg/kg/day for maternal toxicity and embryonic development.

Justification for classification or non-classification

No reproduction toxicity studies are available for DEAE; however suitable data on reproductive organs are available from several valid repeated dose studies (2-year feeding study with rat, a one-year feeding study with Beagle dogs, 14 weeks inhalation study with rats). The data of all these studies indicate that DEAE lacked reproductive toxic properties and did not affect the fertility of the treated animals. Since none of the studies revealed relevantly elevated testis or ovary weights and/or histopathological alterations in those organs which could clearly be attributable to DEAE, the weight of the evidence is that effects on reproduction are also not expected (BAuA Forschungsbericht Fb 984, 2003). Referring to developmental toxicity and teratogenicity, in pregnant rats treated by inhalation during gestation, even the highest test concentration of 0.486 mg/l (486 mg/m3 or 100 ppm), which produced maternally toxic effects, did not lead to adverse developmental effects or teratogenicity. The same applies to pregnant rabbits in which no developmental toxicity has been observed up to and including the highest dose tested. Thus, there is no need for reproduction toxicity classification of DEAE according to the EU Directive 67/548/EEC and to the 1272/2008/EC CLP Regulation.