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Appendices 1-18 to: Report on the Health Effects of Selected Pesticide Coformulants

27   Animal toxicity

27.1   Single dose toxicity

27.1.1   Inhalation

No data have been found.

27.1.2   Oral intake

The oral LD₅₀-values reported for hexamethylenetetramine range from 9200 to higher than 20000 mg/kg b.w. for rats (3 values reported) and 1853 mg/kg b.w. for mice (CIR 1992, IUCLID 2000, JECFA 1974, Loeper & Berzins 1995).

27.1.3   Dermal contact

No data have been found.

27.1.4   Other routes

The LD₅₀-values reported for hexamethylenetetramine range from 215 mg/kg b.w. for mice injected subcutaneously and from 9200 to higher than 10000 mg/kg b.w. for rats injected intravenously. In mice, toxicity symptoms observed after intraperitoneal and subcutaneous administrations were trembling, weakness of the hind quarters, and terminal convulsions. (CIR 1992, IUCLID 2000, JECFA 1974, Loeper & Berzins 1995).

27.2   Repeated dose toxicity

27.2.1   Inhalation

No data have been found.

27.2.2   Oral intake

Groups of 27-102 of each sex of three different strains of mice (CTM, C3Hf/Dp and SWR/Dp) and groups of 48 of each sex of Wistar rats received 0 or 1.0 % hexamethylenetetramine in their drinking water. The calculated daily intake equal to 1 % was 2500 mg/kg b.w. for mice and 1500-2500 mg/kg b.w. for rats according to Loeper & Berzins 1995. The mice were treated for 60 weeks and the rats for 104 weeks. One group of 50 CTM mice per sex was given 0.5 % hexamethylenetetramine for 60 weeks, and another group of 29-50 of each sex received 5 % hexamethylenetetramine for 30 weeks. One group of 12 rats of each sex were given 5 % hexamethylenetetramine for 2 weeks. After the termination of treatment, the animals were observed for the remainder of their lifetimes. Necropsy was performed on all animals, and tissues and lesions taken at necropsy were evaluated microscopically. A yellow discoloration of the furs of treated rats (but not of treated mice) was observed. The SWR mice that received 1 % hexamethylenetetramine and the CTM mice that received 5 % hexamethylenetetramine had a slight retardation of growth. A slight reduction in survival was observed for the CTM mice dosed with 5 % hexamethylenetetramine. The same dose caused 50 % mortality in the rats. Rats from the 5 % group that did not die recovered rapidly and did not have any lasting ill effects. (Della Porta et al. 1968 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 5 or 15 BD II rats of each sex were administered 400 mg per day (equivalent to 1000 mg/kg b.w. per day assuming a rat weight of 400 g) of hexamethylenetetramine by gavage for 90 or 333 days. The only substance related effect was a yellow discolouration of the fur. No macroscopic changes in organ histology or in body weights could be detected. (Brendel 1964 – quoted from CIR 1992, IUCLID 2000, JECFA 1974 and Loeper & Berzins 1995).

Groups of 16 Wistar rats of each sex were fed a standard diet containing either 0 or 0.16 % (equal to an average intake of 0 or 100 mg/kg b.w. per day according to CIR 1992) of hexamethylenetetramine for life. A yellow staining of the hairs of the perineum was observed in one male and three females in the test group. The average life span was 6-9 % longer for controls than for the dosed animals. This difference was associated with slightly lower terminal body weights in the test group, especially for males. No significant differences in body weight, voluntary muscular activity, and relative organ weights of the liver, kidneys, adrenal glands or gonads were observed between the control and treatment group. The majority of deaths in controls as well as dosed rats were attributed to pneumonia. (Natvig et al. 1971 – quoted from CIR 1992and JECFA 1974).

The yellow discolouration of the fur of rats exposed to hexamethylenetetramine orally has been explained as a result of a reaction between formaldehyde present in the urine from treated rats and kynurenine which is a normal constituent of rat hair (Kewitz & Welsch 1966 – quoted from CIR 1992 and IUCLID 2000).

Groups of 2-3 cats of each sex were fed hexamethylenetetramine in the diet at doses of 0 or 50000 ppm (equal to 1250 mg/kg b.w. per day) for 2 years. One female in the dosed group died of a pyrogenic infection of the nasal cavity and paranasal sinuses in the twenty-third month. No differences between control and dosed cats were found in feed consumption, weight gain, appearance, or histology of tissues. (Kewitz 1966 – quoted from CIR 1992 and JECFA 1974).

27.2.3   Dermal contact

Groups of 6 male rabbits had 0 or 2 ml of a 0.20 % solution of hexamethylenetetramine in distilled water applied to the skin for 5 days a week for 6 weeks. The application was not under occlusive patches. General behaviour, hair growth, and weight gain of the rabbits was the same in the control and the test group. No skin irritation was observed. (COLIPA 1989 – quoted from CIR 1992).

Six male rabbits had 0.5 ml of a 0.20 % solution of hexamethylenetetramine in distilled water topically applied to both intact and abraded sites on the flanks for 24 hours under occlusive patches. The rabbits were observed for 72 hours. Slight skin irritation was observed. (COLIPA 1989 – quoted from CIR 1992).

Mild irritation was seen when a 5 % solution of a mixture (ingredients not specified) containing 40 % hexamethylenetetramine was placed on the skin of guinea pigs (DuPont Company 1976 - quoted from Trochimowicz et al. 1993).

27.2.3.1     Sensitisation

The sensitisation potential of hexamethylenetetramine was studied in groups of 5 Dunkin-Hartley albino guinea pigs per sex by performing a guinea pig maximisation test of Magnusson and Kligman. One group was a control group and another group was induced and challenged with hexamethylenetetramine in distilled water at a concentration of 0.20 %. No erythema or oedema was observed in treated animals. (COLIPA 1989 – quoted from CIR 1992).

The sensitisation potential of hexamethylenetetramine was studied in 20 guinea pigs by performing a guinea pig maximisation test. The guinea pigs were induced by intradermal application of 0.1 ml of a 30 % solution of hexamethylenetetramine on the first day followed by epidermal application on day 8 of 0.5 g, which were covered for 48 hours. The guinea pigs were challenged on day 22 by epidermal application of 0.2 ml of a 50 % solution of hexamethylenetetramine, which was covered for 24 hours. Reactions were scored on day 24 and 25. Seventeen of the guinea pigs reacted positive with erythema and swelling. (Degussa AG 1985 – quoted from IUCLID 2000).

The sensitisation potential of AH26 (a root canal filling material containing 25 % hexamethylenetetramine, 10 % silver powder, 60 % bismuth oxide and 5 % titanium dioxide) was studied in groups of 10 female Dunkin-Hartley albino guinea pigs by performing a guinea pig maximisation test of Magnusson and Kligman. One group was a control group and another group was induced intracutaneously with AH26 in saline at a powder/saline ratio of 1.75/1 and percutaneously with AH26 in petrolatum. The animals were challenged with AH26 in petrolatum, 1/10 (w/w). Nine of ten animals were sensitised to AH26. (Kallus et al. 1983 – quoted from CIR 1992 and Loeper & Berzins 1995).

27.2.4   Other routes

27.2.4.1     Intramuscular injection

Groups of 5 BD II rats of each sex were administered 200 mg per day (equivalent to 500 mg/kg b.w. per day assuming a rat weight of 400 g) of hexamethylenetetramine by intramuscular injection for 90 days. The only substance related effect was a yellow discolouration of the fur. No macroscopic changes in organ histology or in body weights could be detected. (Brendel 1964 – quoted from CIR 1992, IUCLID 2000, JECFA 1974 and Loeper & Berzins 1995).

27.2.4.2     Ocular irritation

Six male rabbits had 0.1 ml of a 0.20 % solution of hexamethylenetetramine in distilled water applied once to the conjunctival sac of the eyes. The solution was not rinsed from the eyes. Conjunctival irritancy, iris alterations, or corneal lesions were not observed following dosing. (COLIPA 1989 – quoted from CIR 1992).

Nine New Zealand White albino rabbits had 100 mg of mascara containing 0.1 % hexamethylenetetramine applied to the conjunctival sac of one eye. The eyes of 3 rabbits of each sex were not rinsed after application of the test material while the eyes of 3 male rabbits were rinsed with deionised water 30 seconds after application. The treated eyes of all nine animals were examined 1, 2, 3, 4, and 7 days after application.  The mascara was judged as mildly irritant for unrinsed eyes and non-irritant for rinsed eyes. (Stillmeadow, Inc., 1980 – quoted from CIR 1992).

27.3   Toxicity to reproduction

27.3.1   Inhalation

No data have been found.

27.3.2   Oral intake

Groups of 9-11 female Beagle dogs were fed 0, 600 or 1250 ppm (equal to about 0, 15 or 31 mg/kg b.w. per day based on an average body weight of 12 kg according to JECFA 1974) hexamethylenetetramine on days 4 to 56 after mating. Pregnancy rate, weight gain during pregnancy, length of gestation and litter size was not affected by treatment. In the 1250 ppm group, the percentage of stillborn pups was slightly increased, and the weight gain and the survival to weaning of the pups were slightly impaired. No congenital malformations were observed in any of the 264 live-born and 20 stillborn pups. Some of the pups were kept for observation and a few of them were being used for breeding. After 2 years, all were normal in behaviour, motility, and muscular coordination. Neither the bitches nor the pups had any abnormalities or reproductive disorders. (Hurni & Ohder 1973 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 16 Wistar rats of each sex were fed a standard diet containing either 0 or 0.16 % (equal to an average intake of 0 or 100 mg/kg b.w. per day according to CIR 1992) of hexamethylenetetramine for life. After 3 months of administration, the males and females of the control and test groups were mated. No significant difference was observed in fertility. In both groups, 16 male and 16 female offspring were chosen and fed the same diet as their parents from the time of weaning to natural death. No difference was observed in the relative organ or body weights or in voluntary muscular activity between test and control animals. A yellow staining of the hairs of the perineum was observed in some of the dosed rats. (Natvig et al. 1971 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 12 female and 6 male Wistar rats were given drinking water containing either 0 or 1 % (equal to about 0 or 2000 mg/kg b.w. per day according to Loeper & Berzins 1995) of hexamethylenetetramine starting two weeks before mating. The females were treated during both pregnancy and lactation. No difference were observed between the treated and control groups in regard to fertility, litter size, and malformations. From the pups that were born, 24 of each sex were given 0 or 1 % hexamethylenetetramine in the drinking water until 20 weeks of age. Up to week 9 of age for males and week 20 of age for females, the body weights of the treated animals were significantly lower than the controls. At necropsy, no differences were observed in respect to organ weight, and gross and microscopic changes. (Della Porta et al. 1966, 1970 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 80, 80, or 245 rats were given 0, 5, or 50 mg/kg b.w. per day of hexamethylenetetramine in their drinking water in a five generation study that lasted 3.5 years. At half-yearly intervals starting at 1.5 years, animals (including pregnant dams) were selected from each group to be used for study of lesions. From that time on, no lesions due to hexamethylenetetramine were found in test animals, foetuses, or placenta. No further details were given. (Malorny 1966 – quoted from CIR 1992 and JECFA 1974).

Groups of 10 rats of each sex were fed 0, 400, 800, or 1600 ppm (equivalent to 0, 20, 40, or 80 mg/kg b.w. per day) of hexamethylenetetramine for 2 years. The ten pairs were mated at the age of 20, 28, and 35 weeks. No differences were observed in growth rate, survival, reproduction, offspring viability, or lesions between treated and control groups. No further details were given. (Berglund 1966 – quoted from CIR 1992 and JECFA 1974).

Groups of 1-4 mongrel dogs of each sex were fed 0 or 1250-1875 ppm (equal to about 0 or 94-141 mg/kg b.w. per day according to IUCLID 2000) of hexamethylenetetramine for 32 months. Groups of 2-3 pups of each sex from litters of these dogs were fed 0 or 1250 ppm of hexamethylenetetramine for 22 months. Subsequently, the test animals were fed control diet and the controls were fed the 1250 ppm diet for 1 year. No differences were observed in feed consumption, growth, reproduction, litter number, or litter weight between treated and control groups. Of the 30 litters in the dosed group, approximately 20 had a few stillborn and cannibalised pups, and 5 pups were born with abnormalities. Of the 16 litters in the control group, 1 had stillborn pups, and no pups had malformations. No further details were given. (Kewitz 1966 – quoted from CIR 1992, IUCLID 2000 and JECFA 1974).

27.3.3   Dermal contact

No data have been found.

27.3.4   Other routes

No data have been found.

27.4   Mutagenic and genotoxic effects

27.4.1   In vitro studies

In four studies, hexamethylenetetramine was not mutagenic when tested in 2-5 strains (TA98, TA100, TA1535, TA1537 and TA1538) of Salmonella typhimurium in Ames tests with and without metabolic activation systems (Crebelli et al. 1984, 1985, Orstavik & Hongslo 1984, Andrews et al. 1980 – all quoted from CIR 1992).
In one study, hexamethylenetetramine was reported to be mutagenic towards strain TA98 and TA100 (but not towards strain TA1535, TA1537 and TA1538) without metabolic activation systems (Shimuzu et al. 1985 – quoted from Loeper & Berzins 1995).
After in vitro nitrosation hexamethylenetetramine has been reported to be mutagenic toward strain TA98 and TA100 with and without metabolic activation systems (Crebelli et al. 1984, Andrews et al. 1980 – both quoted from CIR 1992).

Hexamethylenetetramine induced a DNA-damaging effect in the recombination assay using the spores of Bacillus subtilis strains H17 and M45 when tested in the absence of a metabolic activation system. The DNA-damaging effect was decreased in the presence of a metabolic activation system. (Ueno & Ishizaki 1984 – quoted from Loeper & Berzins 1995).Hexamethylenetetramine inhibited the growth of a mutant of Escherichia coli, which is deficient in DNA polymerase. The inhibition of cultures was concluded to be dependent on the degradation of hexamethylenetetramine to formaldehyde. (Fluck et al. 1976, Gillner 1987 – both quoted from Loeper & Berzins 1995).

The number of transformations was dose-dependently and significantly increased when hexamethylenetetramine was tested in the Styles‘ cell transformation assay using baby hamster kidney BHK-21/cl.13 cells in the concentration range of 1 to 10000 mg/ml. The transformation activity was observed at a non-toxic or very weak toxic concentration and it was not dependent on a metabolic activation system. As a comparison, an equal number of transformations and equivalent toxicity was observed with 20 mg formaldehyde/ml and 1000 mg hexamethylenetetramine/ml. (Plesner & Hansen 1983 – quoted from CIR 1992 and Loeper & Berzins 1995).

Hexamethylenetetramine was negative in the mouse lymphoma assay with L5178Y TK+/- cells (Dooley et al. 1985 – quoted from IUCLID 2000).

Hexamethylenetetramine was negative for chromosomal aberrations in human leucocytes but positive in HeLa cells (Roehrborn & Vogel 1967, Balderman & Roehrborn 1967 – both quoted from IUCLID 2000).

27.4.2   In vivo studies

Hexamethylenetetramine caused mutations in larval spermatocytes of Drosophila melanogaster at concentrations, which were higher than those used in medical therapy (concentrations were not stated but for medical therapy doses of 1 g twice daily is recommended according to Lægemiddelkataloget (2001)). The causative agent of the mutagenic effect was formic acid, which was present as an impurity in formaldehyde. (Auerbach 1951, 1977, Stumm-Tegethoff 1964, Nafei & Auerbach 1964 – all quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Hexamethylenetetramine induced dominant lethal mutations in C3H mice which were administered the compound intraperitoneally or orally at a dose of 25000 mg/kg b.w. Doses of 800 to 10000 mg/kg b.w. did not cause dominant lethal mutations. (Balderman et al. 1967, Röhrborn & Vogel 1967 – both quoted from Loeper & Berzins 1995).

In a micronucleus assay in C3H mouse, no clastogenic activity was observed in bone marrow after oral administration of approximately 69, 206 or 618 mg/kg b.w. of hexamethylenetetramine for 1 or 5 days. Bone marrow samples were collected 6, 12, and 24 hours after application of the acute dose and 6 hours after the last exposure in the 5-day study. (Vujosevic et al. 1986 – quoted from IUCLID 2000).

27.5   Carcinogenic effects

27.5.1   Inhalation

No data have been found.

27.5.2   Oral intake

Groups of 7-15 Wistar rats of each sex in three successive generations received 1% hexamethylenetetramine (equal to about 2000 mg/kg b.w. per day according to Loeper & Berzins 1995) in the drinking water up to the age of 40 weeks in the F1 and F2 generations and up to 20 weeks in the F3 generation. Another group of rats received 2 % hexamethylenetetramine in the drinking water, and 16 offspring of each sex were treated for 59 weeks with the same dose. A group of 48 rats of each sex served as a control group. All groups were observed for 2 years after exposure. No evidence of carcinogenicity was found in any of the hexamethylenetetramine treated groups of rats. (Della Porta et al. 1970 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 27-102 of each sex of three different strains of mice (CTM, C3Hf/Dp and SWR/Dp) and groups of 48 of each sex of Wistar rats received 0 or 1.0 % hexamethylenetetramine in their drinking water. The calculated daily intake equal to 1 % was 2500 mg/kg b.w. for mice and 1500-2500 mg/kg b.w. for rats according to Loeper & Berzins 1995. The mice were treated for 60 weeks and the rats for 104 weeks. One group of 50 CTM mice per sex was given 0.5 % hexamethylenetetramine for 60 weeks, and another group of 29-50 of each sex received 5 % hexamethylenetetramine for 30 weeks. One group of 12 rats of each sex were given 5 % hexamethylenetetramine for 2 weeks. After the termination of treatment, the animals were observed for the remainder of their lifetimes. Necropsy was performed on all animals, and tissues and lesions taken at necropsy were evaluated microscopically. No evidence of substance related carcinogenicity was found in any of the hexamethylenetetramine treated groups of animals. (Della Porta et al. 1968 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

A total of 80, 80, or 245 rats were given 0, 5, or 50 mg/kg b.w. per day of hexamethylenetetramine in their drinking water in a five generation study that lasted 3.5 years. At half-yearly intervals starting at 1.5 years, animals (including pregnant dams) were selected from each group to be used for pathological studies. From that time on, no changes due to hexamethylenetetramine were found in test animals, foetuses, or placenta. Tumours were observed in three of 48 animals in the high dose group. No further details were given. (Malorny 1966 – quoted from CIR 1992 and JECFA 1974).

Groups of 30 NMRI/Han albino mice of each sex were fed 0 or 1 % of hexamethylenetetramine (equivalent to 1500 mg/kg b.w. per day) in the feed for 2 years. Twenty neoplasms were found in the dosed group and 11 in the control group. With the exception of one control male and two dosed males, all neoplasms occurred in females. Most of the malignant tumours were subcutaneous carcinomas and adenocarcinomas. The author concluded that the possibility of an increased tumour incidence effect by hexamethylenetetramine could not be ruled out. A further study was performed using groups of 50 female mice, which were administered hexamethylenetetramine at concentrations of 0, 0.1, 0.5, or 1 % in the diet. After 31 weeks, no difference in neoplasm incidence was observed between the groups. (Kewitz 1966 – quoted from CIR 1992 and JECFA 1974).

27.5.3   Dermal contact

Groups of 13 mice were treated daily by cutaneous application of chloroform or 10 % hexamethylenetetramine in chloroform for 300 days. No malignant tumours were found in any group. (Kewitz 1966 – quoted from CIR and JECFA 1974).

39-44 CTM mice of each sex and 20 Wistar rats of each sex were injected subcutaneously 5 times every other day with 5 g/kg b.w. of a 30 % solution of hexamethylenetetramine in water. The animals were observed for 100-104 weeks. Necropsy was performed on all animals, and tissues and lesions taken at necropsy were evaluated microscopically. No evidence of substance related carcinogenicity was found in any of the hexamethylenetetramine treated groups of animals. (Della Porta et al. 1968 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 7-15 albino rats of each sex were injected subcutaneously with 1 ml of a 40 % solution of hexamethylenetetramine or with sodium chloride or sucrose (negative controls) weekly for 1½ year. In the dosed rats, 1 injection site spindle-cell sarcoma, 1 distal spindle-cell sarcoma, 1 alveolar mammary carcinoma, 1 fibrosarcoma, and 2 benign tumours were seen. In the control group, 1 blastoma was found in a sodium chloride injected animal. (Kewitz 1966 – quoted from JECFA 1974).

27.5.4   Other routes

No data have been found.

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