chloromethane; methyl chloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

310 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score: 2) study, which is sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Inhalation is the only significant route of exposure for chloromethane.

A two-generation reproduction study in male and female Fischer 344 rats was carried out at chloromethane concentrations of 0, 150, 475 or 1500 ppm (Hamm et al., 1985). The F0 generation (40 males and 80 females per exposure group) was exposed for 10 weeks and during a 2-week mating period (6 h/day, 5days/week and 6 h/day, 7days/week, respectively). A similar exposure protocol was used for the F1 generation, with the exclusion of the highest dose. Exposures to 1500 ppm resulted in sterility (decreased spermatogenesis) that was consistent with severe testicular degeneration (10/10) and epididymal granulomas (3/10) in male rats after 12 weeks. Significantly fewer litters were born to unexposed females mated to males in the 475 ppm dose group. Litter size, sex ratio, pup viability, pup survival and pup growth were not affected by chloromethane in the 475 and 150 ppm dose groups. A trend towards decreased fertility was also found in the 475 ppm dose group in the F1 generation.

In a 2-year inhalation study testicular lesions consisting of diffuse degenerations of the seminiferous tubules were also observed after exposure to chloromethane at 1000 ppm in mice and rats (CIIT, 1981).

Chapin et al. (1984) investigated the development of lesions induced in testes and epididymis and any associated effects on reproductive hormones in F-344 rats after exposure to 3500 ppm chloromethane (6 h/day for 5 days, not exposed for 3 days and exposure again for 4 days). In general, lesions (delay in spermiation, germinal epithelial vacuolization, bilateral epididymal granulomas) seen in animals at day 19 were of higher severity than those seen earlier. In animals killed 70 days following exposure, 70-90% of the seminiferous tubules lacked any germinal cells and varying degrees of recovery of spermiation were observed in 10-30% of the tubules. Based on testosterone and sulfhydryl analysis, the authors concluded that the initial testicular effects of chloromethane are directed at either the late stage spermatidis or the Sertoli cells with a resultant delay in spermiation.

In a dominant lethal assay, Fischer 344 rats were exposed to 0, 1000 and 3000 ppm chloromethane for 5 days. An increase in post-implantation losses could be detected in animals exposed to 3000 ppm (Working et al., 1985a). An increase in pre-implantation losses was observed in rats in both dose groups. The cause of pre-implantation losses was further investigated by Working and Bus (1986) with the result that these losses were likely due to a failure in fertilization rather than to an increase in embryonal deaths. The effects of chloromethane exposure on sperm quality and histopathology in male Fischer rats were investigated in more detail by Working et al. (1985b). Observations made at the 3000 ppm level included significant decreases in testicular spermatid head counts, delay in spermiation, epithelial vacuolization, luminal exfoliation of spermatogenic cells, and multinucleated giant cells as already reported by Chapin et al. (1984). Further, sperm isolated from the vasa deferentia had significantly depressed number and an elevated frequency of abnormal sperm head morphology by week 1 post exposure and significantly depressed sperm motility and increased frequency of headless tails by week 3 post exposure. These changes were all within or close to the normal range by week 16 post exposure.

In conclusion, testicular lesions and epididymal granulomas followed by reduced sperm counts and sperm quality lead to reduced fertility as well as complete infertility in rats. A NOAEC of 150 ppm (310 mg/m³) was identified from the two-generation study of Hamm et al. (1985).

 

 

Critical assessment of data on fertility

Dekant and Colnot (2013) assessed recently for the members of the Methyl chloride REACh consortium the data on fertility.

Dekant and Colnot (2013) reviewed based on the available data, that a specific, secondary effect on sperm of rats has been demonstrated following repeated exposures to high concentrations of chloromethane. In a two-generation reproductive toxicity study, male rats exposed by inhalation to 475 ppm and above were less fertile than controls. In high-dose (1500 ppm) F0-generation males sacrificed immediately after 12 weeks of exposure, treatment-related lesions consisting of minimal to severe atrophy of the seminiferous tubules (10/10 males examined) and granulomas in the epididymis (3/10) were observed. Severely affected tubules were lined by Sertoli cells and by occasional stem cell spermatogonia. In the less affected tubules, decreased numbers of spermatogonia, primary spermatocytes, and/or secondary spermatocytes were found. A LOAEL of 475 ppm and a NOAEL of 150 ppm were identified in the two-generation study.

At high exposure concentrations (1000 to 5000 ppm), studies in rats have demonstrated that chloromethane results in reduced fertility, testicular toxicity (seminiferous epithelium degeneration, delayed spermiation, reduced testicular weight and numbers of sperm and spermatids, sperm abnormalities and reduced motility, abnormal histopathology, reduced levels of non-protein sulfhydryl content (NPSH) and circulating testosterone), epididymal toxicity (inflammation, sperm granulomas, reduced NPSH levels) and dominant lethal effects. Degeneration and atrophy of the seminiferous tubules were also observed in the testes of mice at 1,000 ppm during a lifetime-study. Pre- and post-implantation losses observed in dominant lethal assays in rats are also likely due to cytotoxicity to sperm and mutations caused by epididymal inflammation.

Embryonic or fetal loss occurring after implantation (post-implantation loss) observed in dominant lethal tests has been attributed to DNA-damage caused indirectly by epididymal inflammation, rather than to direct genotoxic effect. The collective data, including studies with the anti-inflammatory agent BW755C, which inhibits chloromethane-induced epididymal inflammation and post-implantation loss, but not testicular toxicity or preimplantation loss, strongly support the conclusion that the preimplantation loss results from the cytotoxic effects of chloromethane on sperm located in the testes, with consequent failure of fertilization due to low sperm number and poor sperm quality.

Dekant and Colnot (2013) summarized, that the available data strongly support the conclusion, that chloromethane-induced reproductive toxicity is caused by thresholded modes of action and not by genotoxicity.

Moreover, Dekant and Colnot (2013) addressed the question, if chloromethane might be an “endocrine disruptor”. Since chloromethane causes effects on fertility, it may be argued that these effects are due to adverse effects on the endocrine system. Chemicals inducing effects on the endocrine system are also termed “endocrine disruptors” by some stakeholders. According to the most widely accepted scientific definition, “an endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub)populations” (WHO/IPCS, 2002). Thus, this definition embodies two key criteria for identifying an “endocrine disruptor”: specific interference with the function of the endocrine system and biologically meaningful adverse effects in an intact organism caused by this endocrine interference. It is important to note in this context that endocrine disruption is not an endpoint per se, but a mechanism/mode of action (Kortenkamp et al., 2011; Marx‐Stoelting et al., 2011).

Potential mechanisms for chloromethane-induced toxicity to the testes were experimentally addressed. In male F-344 rats, inhalation of toxic concentrations of chloromethane (3,500 ppm) for five consecutive days (6 h/day) resulted in a decrease of circulating levels of testosterone from 120 ± 31 ng/mL in controls to < 6 ng/mL in treated animals. However, when challenged with human chorionic gonadotropin (hCG) to evaluate Leydig cell function, or luteinizing hormone releasing hormone (LHRH) to evaluate pituitary function, chloromethane-exposed and control rats responded similarly in terms of induced serum testosterone levels, suggesting that both the pituitary and Leydig cells were relatively unaffected by chloromethane. The authors postulated that chloromethane may rather act in the brain to lower serum testosterone (Chapin et al., 1984). Furthermore, the effect on testosterone proved to be transient, as the concentration of circulating testosterone in the serum recovered to detectable concentrations 48 h later, but remained lower than the control for between 2 and 3 weeks post exposure (Working et al., 1985). There are several shortcomings associated with this study complicating the interpretation of its findings.

A previous study in F‐344 rats using the same exposure conditions identified the lowest tested concentration of 2,000 ppm as a LOAEC, with 100% of the animals showing testicular atrophy and renal tubular degeneration (Chapin et al., 1984; IPCS, 2000). Higher concentrations additionally produced significant clinical effects and resulted in adrenal, hepatic and cerebellar lesions. Thus, under the selected experimental conditions, the concentration used by Chapin et al. 1984 (3,500 ppm) likely produced overt toxicity in F‐344 rats (no information included).

Concerning the decrease in serum testosterone concentrations described, the interpretation of the data is also hampered by the fact that blood samples were taken from only four animals which is inconclusive since significant variations in testosterone levels are described in laboratory rats sampled under typical laboratory conditions (Heywood, 1980). A significant effect on serum testosterone was observed only after five consecutive days of exposure to 3,500 ppm. However, as reported in Working et al., testosterone levels recovered during the following days without exposures. Testicular lesions were observed starting from day 7. The causal relation of transiently reduced testosterone levels (measured on day 5 only) with testicular damage is therefore questionable.

While chloromethane at high, toxic concentrations may interfere with hormonal balance in male rats, this endocrine effect is most likely not the cause for the observed effects on sperm quality and fertility. As summarized above, preimplantation loss has been attributed to glutathione depletion in testis and epididymis, resulting in direct toxic effects on sperm located in the testes, with consequent failure of fertilization due to low sperm number and poor sperm quality. The conclusion that chloromethane‐induced transient decrease of testosterone has little role in causing the marked testicular toxicity and prolonged effects on sperm quality is additionally supported by studies performed with the structural analogue bromomethane. Bromomethane‐induced decreases in hormone concentration did not have any lasting effect on spermatogenesis or sperm quality (Hurtt and Working, 1988).

Therefore, the effects of chloromethane on sperm quality and fertility are due to a direct toxicity on the testes and not mediated by adverse changes in hormone concentrations. Thus, chloromethane is not an “endocrine disruptor” as defined by WHO/IPCS (2002).

 

 

References:

Chapin, R.E. et al. (1984) Studies of lesions induced in the testis and epididymis of F-344 rats by inhaled methyl chloride. Toxicology and Applied Pharmacology, 76(2):328-343 [as cited in: OECD SIDS Chloromethane, 2004]

Dekant W. and Colnot, T. (2013) Expert Review: Can the German OEL-value (MAK) for Chloromethane ( chloromethane) be considered to be equivalent to a DNEL within the REACH-framework?

Hurtt, M. E. and Working, P. K. (1988). Evaluation of spermatogenesis and sperm quality in the rat following acute inhalation exposure to methyl bromide. Fundamental and Applied Toxicology. 10, 490‐498 [as cited in: Dekant and Colnot, 2013].

Kortenkamp, A., Martin, O., Faust, M., Evans, R., McKinlay, R., Orton, F., Rosivatz, E. (2011). State of the Art Assessment of Endocrine Disrupters. Final Report. Available at:http://ec.europa.eu/environment/endocrine/documents/4_SOTA%20EDC%20Final%20Report%20V3%206%20Feb%2012.pdf [Last accessed 16/11/2012] [as cited in: Dekant and Colnot, 2013].

Marx‐Stoelting, P., Pfeil, R., Solecki, R., Ulbrich, B., Grote, K., Ritz, V., Banasiak, U., Heinrich‐Hirsch, B., Moeller, T., Chahoud, I., and Hirsch‐Ernst, K. I. (2011) Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans. Reproductive Toxicology. 31, 574‐584 [as cited in: Dekant and Colnot, 2013].

WHO/IPCS (2002). Global assessment of the state‐of‐the‐science of endocrine disruptors. Eds: Damstra, T., Barlow, S., Bergman, A., Kavlock, R. and Van der Kraak, G., WHO/PCS/EDC/02.2, World Health Organisation, Geneva. 180 pp [as cited in: Dekant and Colnot, 2013].

Working, P. K., Bus, J. S., and Hamm Jr, T. E. (1985). Reproductive effects of inhaled methyl chloride inthe male Fischer 344 rat: II. Spermatogonial toxicity and sperm quality. Toxicol. Appl. Pharmacol. 77, 144‐157 [as cited in: Dekant and Colnot, 2013].

Working, P.K. and Bus, J.S. (1986). Failure of fertilisation as a cause of preimplantation loss induced by methyl chloride in Fischer 344 rats. Toxicology and applied pharmacology, 86:124 -130 [as cited in: US EPA, Toxicological review of methyl chloride, 2001].

 

 

Short description of key information:
NOAEC (rat) = 310 mg/m³ (Hamm et al., 1985)

Justification for selection of Effect on fertility via inhalation route:
The selected study is the most adequate and reliable study with the lowest dose descriptor.

Effects on developmental toxicity

Description of key information

NOAEC (rabbit, teratogenicity) ≥ 2086 mg/m³ (Theuns-van Vliet, 2016)
NOAEC (rabbit, maternal toxicity) ≥ 2086 mg/m³ (Theuns-van Vliet, 2016)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 086 mg/m³

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Chloromethane (MeCl) has been tested for developmental toxicity in mice, rats and rabbits (see Table 1).

Table 1: Developmental toxicity in mice rats and rabbits

Species	Concentration (ppm)	Observations	Authors	Research institute
Mice	0, 100, 500, 1500	Heart effects (see below)	Wolkowski-Tyl et al. (1983a)	CIIT
Mice	0, 250, 500, 750	Heart effects (see below)	Wolkowski-Tyl et al. (1983b)	RTI/CIIT
Mice	0, 250/300 ppm (24 h) 0, 1000 ppm (12 h)	No developmental toxicity	John-Greene et al. (1985)	CIIT
Rats	0, 100, 500, 1500 ppm	No developmental toxicity	Wolkowski-Tyl et al. (1983a)	CIIT
Rabbits	0, 250, 500, 1000 ppm	No developmental toxicity	Theuns-van Vliet (2016)	TNO Triskelion

RTI = Research Triangle Institute, Research Triangle Park, NC 27709, USA; CIIT = Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709, USA;

 

Studies in mice

Wolkowski-Tyl et al. (1983a) exposed groups of 33 pregnant mice to 100, 500 and 1500 ppm MeCl during gestation days 6-17 (the mice were female C57BL/6 mice bred to C3H males producing B6C3F1 offspring). Due to severe toxicity, the mice of the 1500 ppm group were prematurely sacrificed between gestation days 10-14. Upon necropsy, these animals showed necrosis of the neurons in the internal granular layer of the cerebellum. It was stated that in the mice of the other groups no maternal toxicity was observed but the parameters tested only consisted of determination of food and water intake and body weight gain. No reproductive parameters were significantly affected by MeCl. Fetal body weight and fetal crown-rump length were slightly, but not statistically significantly, increased. No treatment-related external and skeletal effects were observed. Visceral examination revealed small, but statistically significant, increases in the incidence of a heart anomaly in the 500 ppm group. The lesion involved a reduction or absence of the atrioventricular valves, chordae tendineae, and papillary muscles in 6 of 17 litters was distributed between left side (bicuspid or mitral valve) in 3 fetuses and right side (tricuspid valve) in 6 fetuses.

 

So this study in mice showed that 3 fetuses in the 500 ppm group had left heart side underdeveloped and 6 fetuses the right heart side, thus 9 in total, which according to the authors corresponds to 16.65 ± 7.14%. The defects were summarized as a reduction or absence of the atrioventricular valves, reduction or absence of chordae tendinae, and reduction or absence in papillary muscles, without giving any details which was seen in which animal. The authors concluded that 500 ppm was teratogenic in mice. 

 

To further explore this effect, mated female mice (74-77/group; the mice were female C57BL/6 mice bred to C3H males producing B6C3F1 offspring) were exposed to MeCl (0, 250, 500 and 750 ppm, 6 h/day from gestation day 6-17 and sacrificed on gestation day 18 (Wolkowski-Tyl et al., 1983b). From the seventh day of exposure (day 12 of gestation), females exposed to 750 ppm displayed ataxia, tremors, convulsions and hypersensitivity to touch and sound. Six females in this group died and one was killed in extremis. Only the survivors in this group showed a statistically significantly decreased body weight gain on gestation day 18, and a reduced absolute weight gain (weight gain minus gravid uterine weight). No treatment-related effects were observed on pregnancy rate, gravid uterine weight, maternal liver weight, numbers of implantations, resorptions, dead fetuses, live fetuses, sex ratio, mean fetal body weight. Fetuses were examined for external and visceral abnormalities. The authors reported a statistically significant and concentration-related effect on the incidence of affected fetuses. All but one malformation (an umbilical hernia in the 250 ppm group) were observed in the heart. However, the only statistically significant change at 500 ppm (Chi-square test) consisted of an increase in the number of fetuses malformed (11/444 versus 3/433 in controls) but not in the percentage fetuses malformed (2.5% versus 0.69% in controls) and also not in the number or percentage of litters with malformations at 500 ppm. No effects were observed at 250 ppm. The authors indicated that abnormalities of the heart were observed in the 500 and 750 ppm groups and that these included reductions in the number of chordae tendineae and papillary muscles, abnormal tricuspid valve, globular heart, white spots in left ventricle, small right ventricle.

 

The authors described several different heart effects as if these were all seen in a concentration-related way. However, these findings were either of very low incidence, seen in single animals and/or were scattered amongst the groups (including controls) except for one observation, i.e. a reduction in the number of papillary muscles (in bold).

 

Table 2. Observations in the hearts of mouse fetuses (Wolkowski-Tyl et al., 1983b)

 

Concentration (ppm) (number of fetuses)	0  (433)	250 (458)	500 (444)	750 (400)
Reduced number of chordae tendinae on right	-	2	-	-
Abnormal tricuspid valve	-	1	-	1
Globular heart	1	1	2	1
Reduced number of papillary muscles on right	2 (0.46%)	2 (0.44%)	7 (1.58%)	14 (3.50%)
White spots in left ventricle	-	-	2 (0.45%)	4 (1.0%)
Small right ventricle	-	-	1	1

 

So based on the two studies the only corresponding effect would be an effect on papillary muscles, with in the 2nd study an incidence of 1.58% fetuses at 500 ppm and 3.50% at 750 ppm, but also importantly, an incidence of 0.46% in the control group, indicating that this effect also occurs in controls. It is not clear what % this would have been in the first study as 3 effects were mentioned together (reduction or absence of the atrioventricular valves, chordae tendinae, papillary muscles). In the 2nd study, the chordae tendinae effect and the abnormal heart valve were seen in only 1-2 animals and were not concentration-related. The calcinated white spots could not have been observed in the first study because of technical reasons but were seen at a very low incidence in the second study and are possibly of a spontaneous nature. Thus except for the effect on papillary muscles, the two studies seemed to show quite variable results. In addition, the first study described a ‘reduction or absence in papillary muscles’ whereas the second study described a ‘reduction in the number of papillary muscles’.

 

In addition, although MeCl has been reported as causing cardiac malformations in mice, these findings are tempered by significant concerns associated with both the validity of the methods used to identify the putative lesions, and the ultimate impact of those methods on the replicability of these findings across multiple studies and investigators. John-Greene et al. (1985; also CIIT) challenged the ability of methods used to identify subtle cardiac malformations reported in Wolkowski-Tyl et al. (1983a,b), and noted that as experience was gained in fetal evaluation in the laboratory that conducted the original study, and introducing the requirement of evaluation the fetal responses with investigators blinded to treatment, cardiac malformations were no longer evident. This conclusion regarding concerns for the replicability of the fetal examinations is in part supported in the summary data presented by Wolkowski-Tyl (1985) in which fetal responses reported in the first study (Wolkowski-Tyl et al., 1983a) also did not replicate across a common concentration level when examinations were conducted in different laboratories. The statistically significant 16.65% incidence of percent fetuses/litter affected following 500 ppm MeCl exposure in the first study was not statistically elevated in a second study using this same exposure concentration (3.48% fetuses/litter affected; controls 0.68% fetuses/litter affected; Wolkowski-Tyl et al, 1983b). In addition, a higher 750 ppm MeCl exposure used in the second study resulted in a response of 5.12% affected fetuses/litter that was substantially less than the 16.65% lesion incidence per litter reported in the first study at the lower concentration of 500 ppm. Also, the 750 ppm exposure was associated with significant maternal toxicity including tremors, convulsions and increased mortality, questioning the usefulness of this concentration for hazard evaluation. The high concentration maternal toxicity noted at 750 ppm is entirely consistent with the mode of action identified for systemic toxicity associated with high concentration exposures to MeCl. 

 

Chellman et al. (1986) reported that pretreatment of male B6C3F1 mice with the glutathione depleting agent buthionine sulfoximine protected against high concentration (2500 ppm MeCl) induced mortality and associated CNS toxicity (tremors, ataxia, forelimb/hindlimb paralysis), and attributed the protection to blocking formation of putative glutathione-conjugate derived toxic metabolite(s) which is considered a key step in the metabolism of MeCl.

 

In a similar experiment as Wolkowski-Tyl et al. (1983ab), John-Greene et al. (1985) exposed pregnant C57BL mice, carrying B6C3F1 mice, on day 11.5 to 12.5 (considering the presence of a copulatory plug as day 0; day 11.5-12.5 considered the critical period for development of the embryonal heart) to 250 or 300 ppm MeCl. In addition, pregnant mice were exposed from day 11.5 to 12.0 to 1000 ppm MeCl. No treatment-related heart lesions were observed after ‘blind’ fetal examinations. The authors concluded that there was a considerable inter-animal variability in the appearance of the papillary muscles of the heart and the inherent difficulty in confirming their presence owing to their small size and the delicate and precise dissection required to view them.

 

Wolkowski-Tyl (1985) on her turn, questioned whether the inability of John-Greene et al. (1985) to replicate her earlier studies might have been due to differences in the testing protocols, and specifically noted that the period of gestation examined by John-Greene and co-workers, gestation day 11.5-12.5, might not have encompassed the full gestational period of cardiac development.

 

Importantly, however, John-Greene et al. (1985) noted that a pilot study using the same technician who had made the original diagnosis in Wolkowski-Tyl et al (1983a) initially identified increased cardiac malformations using the abbreviated gestational exposure protocol. The inability of subsequent experiments conducted by John-Greene and co-workers to replicate the cardiac defects suggests that the initial pilot findings of John-Greene et al. (1985) as well as those of Wolkowski-Tyl et al. (1983ab) were indeed due to refinements in the examination methods and/or greater experience of the laboratory technician(s) in examining fetal hearts, and thus likely do not represent a true treatment-related finding. These were more likely an artifact of the section technique or a misdiagnosis. Finally, John-Greene et al. (1985) indicated that specific alterations of the papillary muscles in the absence of anomalies of the ventricular septum or great vessels are uncommon in rodents and humans as well.

 

Study in rats

Wolkowski-Tyl et al. (1983a) also investigated structural teratogenicity in pregnant Fischer 344 rats through gestation days 7-19 following the same exposure regimen as the mice. In the highest concentration group maternal toxicity (reduction in body weight and food consumption) occurred and a statistically significant reduction in fetal body weight and female fetal crown-to-rump length was seen. In addition, there were indications of skeletal immaturities such as reduced ossification. No teratological malformations were observed. On the basis of these results the NOAEC for maternal toxicity was set at 500 ppm and the NOAEC for teratogenicity was at least 1500 ppm.

 

Study in rabbits

In a recent performed OECD 414 GLP study (Theuns – van Vliet, 2016), New Zealand White rabbits were exposed to test atmospheres in whole body exposure chambers for 6 hours per day at concentrations of 250, 500 or 1000 ppm MeCl (corresponding to 546, 1053 and 2086 mg/m³). Each group comprised 22 mated animals and in-life parameters included mortality and morbidity, body weight and food consumption. At gestation day 29 caesarean section was performed and necropsy parameters included examination of the dams for gross anatomical changes, uterus and ovary weight. The number and distribution of implantation sites, live and dead fetuses and resorptions were recorded. In addition, placentas and fetuses were weighed individually. Fetuses were examined for external and visceral malformation. Fetal hearts were examined in situ according to Staples (1974). After visceral examination the fetal bodies were processed and stained with Alzarin Red S. for skeletal examination. Heads of half of the fetuses in each litter were fixed in Bouin’s fixative for visceral head examination. Based on the absence of clinical signs and effects on feed intake and only a slight and transient effect on body weight in the high concentration group, the No-Observed-Adverse-Effect-Level (NOAEC) for maternal toxicity was set at 1000 ppm. Based on the absence of exposure related effects on the mean number of implantation sites, early and late resorptions, live fetuses, fetus weight and fetal external, visceral and skeletal observations, the NOAEC for developmental toxicity was set at 1000 ppm.

 

References

Chellman GJ, White RD, Norton RM, Bus JS (1986) Inhibition of the acute toxicity of methyl chloride in male B6C3F1 mice by glutathione depletion. Toxicol. Appl. Pharmacol. 86, 93-104

Dutch Health Council (2004) Methylchloride – Evaluation of the effects on reproduction, recommendation for classification, No. 2004/10OSH, the Hague, the Netherlands, Sep 9

John-Greene JA, Welsch F, Bus JS (1985) Comments on heart malformations in B6C3F1 mouse fetuses induced by exposure to Methyl Chloride – continuing efforts to understand the etiology and interpretation of an unusual lesion. Teratol. 32, 483-487

Staples RE (1974) Detection of visceral alterations in mammalian fetuses. Teratol. 9, 37-38A

Theuns–van Vliet JG (2016) A prenatal development study in New Zealand White rabbits with Methyl Chloride by inhalation preceded by a range finding study. TNO Triskelion, Zeist, the Netherlands

Wolkowski-Tyl R, Phelps M, Davis JK (1983a) Structural teratogenicity evaluation of methylchloride in rats and mice after inhalation exposure. Teratol. 27, 181-195

Wolkowski-Tyl R, Lawton AD, Phelps M, Hamm TE jr (1983b) Evaluation of heart malformations in B6C3F1 mouse fetuses induced by in utero exposure to methylchloride. Teratol. 27, 197-206Wolkowski-Tyl R (1985) Response to comments on heart malformations in B6C3F1 mouse fetuses induced by in utero exposure to methylchloride – continuing efforts to understand the etiology and interpretation of an unusual lesion. Teratol. 32, 489-492

 

Critical assessment of data on developmental toxicity

 

At a meeting of the Working Group “Classification and labelling of dangerous substances” in 1987 “[…], the majority agreed that the questionable heart defects found in mouse embryos do not justify classification as teratogen.” (cited form the minutes of the meeting 1987 in Brussels).

Based on the studies in rats and mice mentioned above the Dutch Health Council (2004) recommended no classification for MeCl with respect to developmental toxicity because of a lack of appropriate data.

 

Based on the data in mice, the consortium decided in 2010 to self-classify MeCl according to Regulation (EC) No 1272/2008 for developmental toxicity Category 2 by following the precautionary principle. In view of the overall toxicity profile it took the position at that time that any further clarification by additional animal experiments would be ethically questionable.

 

In view of the ongoing CoRAP activities Prof. Dr. Wolfgang Dekant was asked for his opinion regarding classification of reproductive toxicity. In view of the remaining doubts on the relevance of the heart effects in mice he concluded that “[…] MeCl is a borderline case for no classification/classification in Hazard Category 2” but “[...] should prudently be considered a potential developmental toxicant” (Dekant et al., 2013).

 

However, the member state committee did not agree that the existing data base with rats and mice is sufficient for the evaluation of developmental toxicity and requested an OECD 414 study in non-rodents (rabbits) to clarify the mode of action of MeCl.

 

This study carried out in 2015, showed that exposure of rabbits to 0, 250, 500 or 1000 ppm did not show developmental toxicity. The hearts of these rabbits were examined according to Staples (1974) as required according to OECD 414.

 

Including the new data with a third species into the hazard assessment for developmental toxicity the following can be concluded:

 

1.           The normal anatomy of the tiny papillary muscles in the mouse heart and the variability of their appearance as observed by John-Greene et al. (1985) complicate a diagnosis. It should also be noted that the technique used to examine the visceral tissue changes in mice is based on Staples (1974), but the OECD testing guideline recommends this method only for non-rodents.

2.           It was noted that MeCl showed much higher toxicity in mice than in rats and rabbits. Rats showed some maternal toxicity at 1500 ppm (not at 500 ppm), and rabbits showed only slight toxicity at 1000 ppm (and not at 500 ppm). Mice, in contrast, in the 1st study had to be killed at 1500 ppm, and showed severe toxicity at 750 ppm in the 2nd study. Thus, especially at higher concentrations mice seem to be more susceptible, which may be due to a difference in metabolism at higher concentrations (see above).

3.           Although it was claimed by Wolkowski et al. (1983b) that a concentration of 500 ppm MeCl showed no maternal toxicity in mice based on determination of food and water intake and body weight gain, sub-clinical toxicity may have influenced fetal growth. Consulting experts on reproduction/developmental toxicity indicated that the kind, number and severity of the heart effects observed in these mice, if not an artifact due to the large intra-animal variability and the difficulty to perform these examinations in mice, could also have been related to a slight retardation in development, which probably would have been recovered during postnatal development.

4.           MeCl is likely to have species specific metabolic characteristics making it difficult to define the best animal model. Therefore, in 2010 it was prudent to propose a classification based on conflicting data in rats and mice notwithstanding the doubts expressed by various experts regarding the relevance of the mouse data (John-Greene et al., 1985; Dutch Health Council, 2004; Dekant and Colnot, 2013). It has also to be strengthened that the mouse strain that showed the developmental heart effects is not commonly used for developmental toxicity testing complicating the interpretation of the relevance also in view of the low incidences of the effects observed. In view of the now available OECD 414 guideline GLP study in a standard non-rodent species and strain (New Zealand White rabbit), the data base for hazard determination has increased which reduces the scientific weight of the mouse data.

Justification for selection of Effect on developmental toxicity: via inhalation route:
The selected study is the most adequate and reliable study.

Justification for classification or non-classification

Chloromethane has not been categorized as a reproductive toxicant in the harmonised classification (Index No. 602-001-00-7) in Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation), Table 3.1 (CLP classification) and Table 3.2 (DSD classification).
 
Fertility:

Based on the experts’ advice (Dekant and Colnot 2013) the consortium decided to self-classify the effects on fertility into category 2 (CLP).
 

Developmental toxicity:

Based on the absence of effects in rats and rabbits as well as the very limited scientific weight of the effects observed in mice chloromethane (MeCl) does not meet the classification criteria for developmental toxiciy.

(Arts, J., Kellert, M., Pottenger, L., Theuns-van Vliet, J., Evaluation of developmental toxicity of Methyl Chloride (Chloromethane) in rats, mice, and rabbits, Regulatory Toxicology and Pharmacology 2019, 103, 274 -281 doi: https://doi.org/10.1016/j.yrtph.2019.02.001)