Registration Dossier

Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Read across data is presented on the "fragments" from which this substance is manufactured :-

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides, CAS Number 68391-05-9, EC Number 269-924-1 and from sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9.

The justification in taking this approach is as follows :-

In the stomach the gastic juice is acidic, made up of acids and enzymes. In such an evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested read-across data from the corresponding quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides is considered approriate in that such substances are likely to dissociate in a similar manner.

Furthermore, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of Quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats

Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)

Group III. The di-and tri-chlorobenzyl substituted

Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g  halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded then it would seem that to consider read-across data from quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the closely structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites to be equally justifiable.

Similarly since the US EPA deem that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is equally justifiable.

Furthermore, in certain organic solvents it has been reported that the exchange constants between nitrite and chloride in quaternary ammonium salts (QAS) are approximately equal. [Zhurnal Analiticheskoi Khimii, 2010, Vol. 65, No. 6, pp. 579–584. (E.M. Rakhman’ko, M.S. Markovskaya, L.S. Stanishevskii, Yu.S. Zubenko, A.R. Tsyganov)]

To that end one study is presented from quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides, CAS Number 68391-05-9, EC Number 269-924-1 and five studies are presented from sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9.

Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides, CAS Number 68391-05-9, EC Number 269-924-1

A study was conducted to evaluate the toxic effects of 13 wk dietary exposure to test material (40% didecyldimethylammonium chloride (DDAC) in water) in rats. The study was conducted according to OECD guideline 408.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was given by dietary admixture toSprague-Dawleyrats for 13 wk at the concentration of 1500, 3000 or 6000 ppm (corresponding to 42, 84 or 175 mg of DDAC/kg/d for the males and 49, 96 or 201 mg of DDAC/kg/d for the females). All animals were monitored for toxic effects including clinical observations, body and organ weights, hematology, serum chemistry, macroscopic and microscopic evaluations.

Under the test conditions, the NOAEL of the test material was determined to be 1500 ppm (corresponding to 42 mg DDAC/kg/d for the males and 49 mg DDAC/kg/d for the females) in rats

Sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9 (1)

In a publication entitiled "Epidemiological and Toxicological Aspects of Nitrates and Nitrites in the Environment" [Shuval HI and Gruener N (1972); Am. J. Public Health 62, 1045-1052) a 2 year study repeat dose (oral) toxicity is reported on sodium nitrite as the primary source.

Under the conditions of this study the NOEL was determined to be 6.7 mg NO2/kg bw/day.

Sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9 (2)

In a NTP study (2001), as a primary source, groups of male and female F344/N rats (10 animals/sex/group) were exposed to 0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 30, 55, 115, 200, or 310 mg sodium nitrite/kg bw/day in males and 0, 40, 80, 130, 225, or 345 mg/kg bw/day in females) in drinking water for 14 weeks.

Under the conditions of the study theNOAELs were not determined although all animals showed methaemoglobin formation.

However, the LOAEL were determined to be :

Males = 115 mg/kg bw/day

Females = 225 mg/kg bw/day

Sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9 (3)

In a NTP study (2001), as a primary source, groups of male and female B6C3F1 mice (10 animals/sex/group) were exposed to 0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 90, 190, 345, 750, or 990 mg/kg  mg sodium nitrite/kg bw/day in males and 0, 120, 240, 445, 840, or 1,230  mg/kg bw/day in females) in drinking water for 14 weeks.

Under the conditions of the study the LOAEL were determined to be :

Males = 750 mg/kg bw/day

Females = 445 mg/kg bw/day

Sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9 (4)

In a NTP study (2001), as a primary source, groups of male and female F344 rats (10 animals/sex/group) were exposed to 0, 750, 1,500, 3,000 ppm sodium nitrite in drinking water (equivalent to average daily doses of approximately 0, 35, 70 or 130 mg sodium nitrite/kg bw/day in males and 0, 40, 80 or 150 mg/kg bw/day in females) in drinking water for 2 years.

Under the conditions of the study theNOAEL were determined to be :

Males = 130 mg/kg bw/day

Females = 150 mg/kg bw/day

Sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9 (5)

In a NTP study (2001), as a primary source, groups of male and female B6C3F1 mice (10 animals/sex/group) were exposed to 0, 750, 1500 or 3000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 60, 120 or 220 mg/kg bw/day for males and 0, 45, 90 or 165 mg/kg bw/day for females) in drinking water for 2 years.

Under the conditions of the study theNOAEL were determined to be :

Males = 220 mg/kg bw/day

Females = 165 mg/kg bw/day

Therefore from the figures available the lowest NOEL/NOAEL to be considered are :-

42 mg/kg bw/d for quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides, CAS Number 68391-05-9, EC Number 269-924-1 and 6.7 mg/kg bw/d for

sodium nitrite, CAS Number 7632-00-0, EC Number 231-555-9. It should be acknowledged that this value for sodium nitrite is a factor of ca. 20 to 30 times lower than the NOAEL derived from the other studies reported.

Furthermore the 38th series of "Reports of the Scientific Committee for Food (1997) as regards "nitrites" states the following :-

The effects of continuous administration of nitrite to experimental animals in drinking water include vasodilation and sedation, methaemoglobinaemia and histopathological changes in cardiac muscle, lung, liver, spleen, kidney and adrenals. Hypertrophy of the adrenal zona glomerulosa is the most sensitive indicator in the rat and since the previous review additional studies have been conducted to clarify the possible role of the potassium ion and to establish a clear NOEL (TiL et al. 1990; Til & Kuper, 1995). In the most sensitive strain of Wistar rat,no significant changes were observed at doses of 10 mg KNO2 /kg b.w. while in a different Wistar strain adrenal changes were only seen at three times higher doses. Similar effects were seen with NaNO2 but with reduced incidence and severity. The mechanism by which nitrite affects the adrenal remains unclear but it has been suggested that it is related to the appearance of methaernoglobinaemia and subsequent adaptation, possibly due to competition for NADPH between methaemoglobin reductase and hydroxylases involved in corticosteroid synthesis (Boink et al.1995). Together with effects of nitrite on urinary steroids (Violanthe et. al. 1973) this suggests an adaptive change with time and explains why adrenal effects were not observed in lons-term studies.

However, given that the "nitrite" functionality present in quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites accounts for only approximately 8.7% of the substance and the "quaternary ammonium compounds, di-C12 -18 -alkyldimethyl fragment" accounts for approximately 91.3% of the substance, it is envisaged that in order for 6.7 mg to be released per day then ca. 70 - 75 mg per day of quaternary ammonium compounds, di-C12 -18 -alkyldimethyl fragment would need to be released which far exceeds its NOEL of 42 mg/kg bw/d.

It is therefore concluded that the NOEL for quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, nitrites should be regarded as 42 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic information given
Justification for type of information:
Since in the stomach the gastic juice is acidic, made up of acids and enzymes, it is suggested that in this evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In addition, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: FDA (21 CFR, Part 58)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Source: Taconic Farms (Gemantown, NY)
Time Held Before Studies:14 days (males) or 15 days (females)
Average Age When Studies Began:7 weeks
Duration of Exposure:14 weeks
Average Age at Necropsy: 20 weeks
Animals were distributed randomly into groups of approximately equal initial mean body weights
Animals per Cage: 5
Method of Animal Identification: Tail tattoo
Diet: NIH-07 open formula powdered diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water: Charcoal-filtered deionized water via amber glass bottles with stainless steel sipper tubes, available ad libitum and changed twice weekly
Cages: Solid-bottom polycarbonate
Bedding: Sani-ChipsCage
Filters: DuPont 2024 spun-bonded polyester filter (Snow Filtration Co., Cincinnati, OH)
Racks: Stainless steelAnimal Room Environment:
Temperature: 72 degree +/-3 degree F;
Relative humidity: 50% +/-15%;
Room fluorescent light: 12 hours/day;
Room air changes: Minimum 10/hour
Route of administration:
oral: drinking water
Vehicle:
water
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
Doses / Concentrations:0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of ca. 30, 55, 115, 200, or 310 mg sodium nitrite/kg body weight to males and 40, 80, 130, 225, or 345 mg/kg to females)
Basis:nominal in water
Dose / conc.:
375 ppm
Remarks:
Equivalent to 30 mg sodium nitrite/kg bw for males and 40 mg/kg bw for females;
Basis: nominal in water
Dose / conc.:
750 ppm
Remarks:
Equivalent to 55 mg sodium nitrite/kg bw for males and 80 mg/kg bw for females;
Basis: nominal in water
Dose / conc.:
1 500 ppm
Remarks:
Equivalent to 115 mg sodium nitrite/kg bw for males and 130 mg/kg bw for females;
Basis: nominal in water
Dose / conc.:
3 000 ppm
Remarks:
Equivalent to 200 mg sodium nitrite/kg bw for males and 225 mg/kg bw for females;
Basis: nominal in water
Dose / conc.:
5 000 ppm
Remarks:
Equivalent to 310 mg sodium nitrite/kg bw for males and 345 mg/kg bw for females;
Basis: nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
Type and Frequency of Observation: twice daily. Core study animals were weighed and clinical findings were recorded initially, weekly, and at the end of the studies. Drinking water consumption was measured daily.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation
Necropsy: Necropsy was performed on all core study. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
Clinical Pathology: Blood for hematology and clinical chemistry was collected from the retroorbital sinus of anesthetized clinical pathology study rats on days 5 and 19 and from core study rats at the end of the study. Two blood samples each were collected from the abdominal aorta of 15 male and 15 female clinical pathology study rats on day 70 (20.00 or 22.00 hours) or 71 (09.00 hours) for hemoglobin, methemoglobin and nitrosamine concentrations; stomach contents were also collected for nitrosamine concentrations. Blood and stomach contents were collected from five males and five females at each time point.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials; erythrocyte and platelet morphologic assessments; methemoglobin concentration; reduced glutathione concentration in erythrocytes; and Heinz body count.
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.
Nitrosamine concentrations: serum and gastric nitrosamine
Histopathology: Complete histopathology was performed on 0 and 5,000 ppm core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, muscle, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, skin, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. The forestomach of 750 (males), 1,500, and 3,000 ppm animals were also examined.
Other examinations:
Sperm Motility and Vaginal Cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology evaluations from male rats in the 0, 375, 1,500, and 5,000 ppm groups and female rats in the 0, 375, 750, and 3,000 ppm groups. The left cauda, epididymis, and testis were weighed. The following parameters were evaluated: spermatid heads per gram testis, spermatid heads per testis, spermatid count, motility, and concentration. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.
Statistics:
Statistical analyses were conducted about survival rate, neoplasm and non-neoplastic lesion incidences.
Details on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
Clinical signs: Brown discoloration in the eyes and cyanosis of the mouth, tongue, ears, and feet of 200 and 310 mg/kg bw/day males and of 130 mg/kg bw/day and higher females.
Bodyweight and food consumption: Body weights of 200 and 310 mg/kg bw/day males and 345 mg/kg bw/day females were significantly less than those of the controls. Water consumption by 310 mg/kg bw/day males and 225 and 345 mg/kg bw/day females was less than that by the controls at weeks 2 and 14.
Mortality and time to death: One 225 mg/kg bw/day female died before the end of the study.
Clinical Pathology: Methaemoglobin levels were significantly elevated in all treated groups compared to the controls by the end of the treatment period. For males, mean methaemoglobin levels after 14 weeks were 0.03±0.01, 0.08±0.01, 0.12±0.02, 0.25±0.07, 0.71±0.20 and 3.38±0.80 g/dL at doses of 0, 30, 55, 115, 200, and 310 mg/kg bw/day. For females, mean methaemoglobin levels after 14 weeks were 0.06±0.02, 0.14±0.02, 0.16±0.02, 0.48±0.05, 0.99±0.20 and 2.27±0.54 g/dL at doses of 0, 40, 80, 130, 225 and 345 mg/kg bw/day.
Haematology: Reticulocyte counts were increased in 200 and 310 mg/kg bw/day males and 225 and 345 mg/kg bw/day females. The erythron was decreased on day 19 but increased by week 14 in 310 mg/kg bw/day males and 345 mg/kg bw/day females.
Gross pathology incidence and severity: The incidences of squamous cell hyperplasia of the forestomach in 310 mg/kg bw/day males and 345 mg/kg bw/day females were significantly increased.Organ weight changes: The relative kidney and spleen weights of 200 and 310 mg/kg bw/day males and 225 and 345 mg/kg bw/day females were significantly greater than those of the controls.
Histopathology: Increased erythropoietic activity in the bone marrow of exposed males and females was observed.
Sperm Motility and Vaginal Cytology: Sperm motility in 115 and 310 mg/kg bw/day males was significantly decreased.
Key result
Dose descriptor:
LOAEL
Effect level:
115 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: decreased sperm motility
Key result
Dose descriptor:
LOAEL
Effect level:
225 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: increased relative weight of the kidney and spleen
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 000 ppm
System:
other: histiocytosis, mastocytosis and sinusal hemorrhage in the mesenteric lymph node
Organ:
lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
3 000 ppm
System:
other: histiocytosis and mastocytosis in the mesenteric lymph node
Organ:
lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm
System:
other: changes in hematological and blood biochemical parameters were recorded
Organ:
lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In a NTP study (2001), as a primary source, groups of male and female F344/N rats (10 animals/sex/group) were exposed to 0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 30, 55, 115, 200, or 310 mg sodium nitrite/kg bw/day in males and 0, 40, 80, 130, 225, or 345 mg/kg bw/day in females) in drinking water for 14 weeks.

The LOAEL were determined to be :
Males = 115 mg/kg bw/day
Females = 225 mg/kg bw/day
Executive summary:

In a NTP study (2001), as a primary source, groups of male and female F344/N rats (10 animals/sex/group) were exposed to 0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 30, 55, 115, 200, or 310 mg sodium nitrite/kg bw/day in males and 0, 40, 80, 130, 225, or 345 mg/kg bw/day in females) in drinking water for 14 weeks.

This study is also referenced in the The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Under the conditions of the study the NOAELs were not determined although all animals showed methaemoglobin formation.

However, the LOAEL were determined to be :

Males = 115 mg/kg bw/day

Females = 225 mg/kg bw/day

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From October 11, 2002 to December 02, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across justification is presented from the structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites.

In the stomach the gastic juice is acidic, made up of acids and enzymes. In such an evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested read-across that data from the corresponding quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides is considered approriate in that such substances are likely to dissociate in a similar manner.

Furthermore, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of Quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA·solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded then it would seem that to consider read-across data from quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides to the closely structurally analogous quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites to be equally justifiable.

Furthermore, in certain organic solvents it has been reported that the exchange constants between nitrite and chloride in quaternary ammonium salts (QAS) are approximately equal. [Zhurnal Analiticheskoi Khimii, 2010, Vol. 65, No. 6, pp. 579–584. (E.M. Rakhman’ko, M.S. Markovskaya, L.S. Stanishevskii, Yu.S. Zubenko, A.R. Tsyganov)]


Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Arquad 2.10-40

- Composition of test material, percentage of components: ca. 40% Didecyldimethylammonium chloride (CAS no.: 7173-51-5)
in water only.
- Batch No.: FP019980246
- Physical state: Colourless to pale yellowish liquid
- Analytical purity: 42.9% active ingredient in water
- Stability: Stable
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Cesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
- Age at study initiation: 6 wk at Day 1 of treatment
- Weight at study initiation: Males: 185 g; females: 153 g
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- Type: Oral via diet in feed
- Vehicle: Plain diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
93 d
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
0, 1500, 3000 and 6000 ppm of test material in diet corresponds to active substance DDAC (males/females): 1500 ppm: 42 /49 mg/kg/d; 3000 ppm : 84/96 mg/kg/d; 6000 ppm : 175 / 201 mg/kg/d
Basis: actual ingested
Dose / conc.:
1 500 ppm
Remarks:
Males, equivalent to 42 mg/kg bw/d
Dose / conc.:
1 500 ppm
Remarks:
Females, equivalent to 49 mg/kg bw/d
Dose / conc.:
3 000 ppm
Remarks:
Males, equivalent to 84 mg/kg bw/d
Dose / conc.:
3 000 ppm
Remarks:
Females, equivalent to 96 mg/kg bw/d
Dose / conc.:
6 000 ppm
Remarks:
Males, equivalent to 175 mg/kg bw/d
Dose / conc.:
6 000 ppm
Remarks:
Females, equivalent to 201 mg/kg bw/d
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, plain diet
Details on study design:
None
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at beginning and end of study

HAEMATOLOGY: Yes
- Number of animals: all animals
- Time points: end of study
- Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Number of animals: all animals
- Time points: end of study
- Parameters: sodium, potassium, chloride, calcium, Inorg. Phosphorous, glucose, urea, total bilirubin, creatinine, total protein, albumin, total cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lipids.
Sacrifice and pathology:
-Organ weights: Yes, all animals
-Organs: adrenals, liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, thyroid, spleen, brain, heart

-Gross and histopathology: Yes, all animals of control and high dose group, animals that prematurely died, and all macroscopic lesions.
Animals of intermediate dose group also investigated in full when possible effects are seen in highest dose group.
-Organs: Macroscopic lesions, Adrenals, Aorta, Brain (including medulla/pons, cerebellar and cerebral cortex), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes with Harderian glands, Femoral bone with articulation, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs with bronchi, Lymph nodes (mandibular and mesenteric), Mammary glands/area, Ovaries (with oviducts), Pancreas, Pituitary gland, Prostate (dorso-lateral and ventral), Rectum, Salivary glands (sublingual and submandibular), Sciatic nerve, Seminal vesicles (including coagulation gland), Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with bone marrow, Stomach with forestomach, Testes, Thymus, Thyroids with parathyroids, Tongue, Trachea, Urinary bladder, Uterus (horns and cervix), Vagina.
Additionally, mesenteric lymph nodes of the low- and intermediate-dose groups were examined.
Other examinations:
-Other examinations: Functional observation battery (FOB)
Statistics:
Depending on normal distribution (Kolmogorov–Lilliefors test, possibly after a Logarithmic transformation of the values – except for organ weights) and following testing of homogeneity of variances between groups: Bartlett test (3 or more groups) or Fisher test (2 groups).
If Homogeneous: Student test (2 groups) or Dunnett test (3 or more groups),
Else: Dunn test (3 or more groups) or Mann–Whitney/Wilcoxon test (2 groups)
If not normal distribution: Dunn test
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
General clinical signs: 6000 ppm: Emaciated appearance in 1/10 male and 2/10 female; Soft feces in 1/10 males and 8/10 females. Further in females: Area of hair loss or scattered hair (6/10) and one female showed a round back. Although 1/10 males given 1500 ppm showed piloerection, no noteworthy clinical signs were observed in any other group over the considered period.
Detailed clinical signs - Except soft feces noted in females at 6000 ppm, the detailed clinical observation had shown no perturbation of the autonomic or physiological functions in any treated group.
- No mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN: Decreased food consumption and body weight gain which were noted in treated males and females at 1500 ppm during the first week of the study, were considered to be related to palatability of the compound in the diet, resulting in a lower food consumption and lower body weight gain. The body weight gain and food consumption of treated animals at 3000 and 6000 ppm was affected by the oral administration (dietary admixture) of DDAC.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): When compared to controls, food consumption of animals treated at 3000 and 6000 ppm was affected during the whole treatment period, with a more marked effect at the highest concentration.
Also in the 1500 ppm group the food intake is decreased during the first treatment week.

OPHTHALMOSCOPIC EXAMINATION: No relevant ophthalmological findings were observed in any group.

HAEMATOLOGY: When compared with control values, a decrease in WBC count was recorded in all treated males and females treated at 6000 ppm. A tendency for lower WBC was observed in treated females at 1500 or 3000 ppm.

CLINICAL CHEMISTRY: - increase in sodium, chloride and inorganic phosphorus plasma levels at 6000 ppm,
- a tendency for decrease in glucose levels in treated groups at 6000 ppm and in females treated at 3000 ppm,
- increase in urea and creatinine plasma levels in all treated male groups, and only a tendency for higher urea values in females treated at 6000 ppm,
- decrease in protein concentration in the 6000 ppm group,
- increase in A/G ratio in males treated at 3000 and 6000 ppm and in females treated at 6000 ppm. This difference could be explained by a decrease in globulins in males. In females, decrease in albumin was noted at 6000 ppm,
- dose-related decreased triglyceride in treated males, a tendency for lower cholesterol values in treated females at 6000 ppm,
- increase in transaminase activities in females treated at 6000 ppm.

ORGAN WEIGHTS: Relative and absolute organ weight differences to control are mostly related to low body weights in 6000 ppm group. Absolute kidney weights are also depressed in mid-dose groups.

GROSS AND HISTOPATHOLOGY: Distended coecum with feces in 9/10 males and all females of the high dose group, and in 4/10 males and 3/10 females of the mid-dose group. Similarly distended colon with faeces was observed in 4/10 males and 2/10 females of the high-dose group.
A brownish colour of the mesenteric lymphnodes was seen in 2/10 males and 4/10 females of the high-dose group, and in one male of the mid-dose group. Histiocytosis and mastocytosis in the mesenteric lymph node were seen at histopathology at and above 3000 ppm.

OTHER FINDINGS
Functional Observation Battery (FOB) - There were no perturbation of either autonomic or physiological functions at any dose-levels.
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight; food consumption; gross pathology; histopathology.
Remarks on result:
other: corresponding to 42 mg DDAC/kg/d for the males and 49 mg DDAC/kg/d for the females
Key result
Dose descriptor:
LOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight; food consumption; gross pathology; histopathology.
Critical effects observed:
not specified

At 6000 ppm, main treatment-related findings were: soft feces, body weight loss (week 1), lower body weight gain and food consumption, perturbation of hematological and blood biochemical parameters, distension of the cecum/colon with feces in all animals, histiocytosis, mastocytosis and sinusal hemorrhage in the mesenteric lymph node, consistent with a continued action of a mild irritant.

At 3000 ppm, body weight gain and food consumption were affected, changes at clinical pathology were noted, the cecum was distended with feces in about third of the animals, histiocytosis and mastocytosis in the mesenteric lymph node were seen at histopathology.

At 1500 ppm, changes in hematological and blood biochemical parameters were recorded and possibly a marginal increase in histiocytosis and mastocytosis in the mesenteric lymph node which all do not immediately seem to be of toxicological significance.

The most obvious effect is the lowering of the food intake caused by the palatability of the compound, which at lower levels improves after some time. Some effects seen in haematology and blood chemistry at the higher dose levels could very well result from a poor alimentary status as opposed to a specific toxic mechanism. At the highest dose level, the finding of distended cecum and/or colon becomes noteworthy, and possibly a higher severity of histiocytosis at microscopic examination, although this aspect is also already observed in controls.

Conclusions:
Under the test conditions, the NOAEL of the test material was determined to be 1500 ppm (corresponding to 42 mg DDAC/kg/d for the males and 49 mg DDAC/kg/d for the females) in rats
Executive summary:

A study was conducted to evaluate the toxic effects of 13 wk dietary exposure to test material (40% didecyldimethylammonium chloride (DDAC) in water) in rats. The study was conducted according to OECD guideline 408.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was given by dietary admixture to Sprague-Dawley rats for 13 wk at the concentration of 1500, 3000 or 6000 ppm (corresponding to 42, 84 or 175 mg of DDAC/kg/d for the males and 49, 96 or 201 mg of DDAC/kg/d for the females). All animals were monitored for toxic effects including clinical observations, body and organ weights, hematology, serum chemistry, macroscopic and microscopic evaluations.

At 6000 ppm, main treatment-related findings were: soft feces, body weight loss (at wk 1), lower body weight gain and food consumption, perturbation of hematological and blood biochemical parameters, distension of the cecum/colon with feces in all animals, histiocytosis, mastocytosis and sinusal hemorrhage in the mesenteric lymph node, consistent with a continued action of a mild irritant.

At 3000 ppm, body weight gain and food consumption were affected, changes at clinical pathology were noted, the cecum was distended with feces in about third of the animals, histiocytosis and mastocytosis in the mesenteric lymph node were seen at histopathology.

At 1500 ppm, changes in hematological and blood biochemical parameters were recorded and possibly a marginal increase in histiocytosis and mastocytosis in the mesenteric lymph node which all do not immediately seem to be of toxicological significance.

Under the test conditions, the NOAEL of the test material was determined to be 1500 ppm (corresponding to 42 mg DDAC/kg/d for the males and 49 mg DDAC/kg/d for the females).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic information given
Justification for type of information:
Since in the stomach the gastic juice is acidic, made up of acids and enzymes, it is suggested that in this evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In addition, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
In a publication entitiled "Epidemiological and Toxicological Aspects of Nitrates and Nitrites in the Environment" [Shuval HI and Gruener N (1972); Am. J. Public Health 62, 1045-1052) a 2 year study repeat dose (oral) toxicity is reported.
GLP compliance:
not specified
Species:
rat
Sex:
male
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Dose / conc.:
0 mg/L drinking water
Remarks:
Equivalent to 0 mg/kg bw/day
Dose / conc.:
100 mg/L drinking water
Remarks:
Equivalent to 10 mg/kg bw/day
Dose / conc.:
1 000 mg/L drinking water
Remarks:
Equivalent to 100 mg/kg bw/day
Dose / conc.:
2 000 mg/L drinking water
Remarks:
Equivalent to 250 mg/kg bw/day
Dose / conc.:
3 000 mg/L drinking water
Remarks:
Equivalent to 350 mg/kg bw/day
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Body weight; once a month
Sacrifice and pathology:
Mortality; Methemoglobin; Blood chemistry; glucose, pyruvate, lactate Pathology; heart, lungs, kidneys, liver, spleen, pancreas, adrenals and some brains.
Details on results:
There were no significant differences in growth, development, mortality or total haemoglobin levels between the control and treated groups. However, the methaemoglobin levels in the groups receiving 100, 250 and 350 mg/kg bw/day sodium nitrite were raised significantly throughout the study and averaged 5, 12 and 22% of total haemoglobin, respectively.The main histopathological changes occurred in the lungs and heart. Focal degeneration and fibrosis of the heart muscle were observed in animals receiving the highest dose of nitrite. The coronary arteries were thin and dilated in these aged animals, instead of thickened and narrow as is usually seen at that age. Changes in the lungs consisted of dilatation of the bronchi with infiltration of lymphocytes and alveolar hyperinflation. Such changes were observed in rats receiving 100, 250 and 350 mg/kg bw/day sodium nitrite.
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: (focal degeneration and fibrosis of the heart, dilatation of the bronchi with infiltration of lymphocytes and alveolar hyperinflation in lungs) equivalent to 6.7 mg NO2/kg bw/day.
Remarks on result:
other: Equivalent to 6.7 mg NO2/kg bw/day
Critical effects observed:
not specified
Conclusions:
In a publication entitiled "Epidemiological and Toxicological Aspects of Nitrates and Nitrites in the Environment" [Shuval HI and Gruener N (1972); Am. J. Public Health 62, 1045-1052) a 2 year study repeat dose (oral) toxicity is reported. The NOEL of sodium nitrite was determined to be 6.7 mg NO2/kg bw/day.
Executive summary:

In a publication entitiled "Epidemiological and Toxicological Aspects of Nitrates and Nitrites in the Environment" [Shuval HI and Gruener N (1972); Am. J. Public Health 62, 1045-1052) a 2 year study repeat dose (oral) toxicity is reported on sodium nitrite as the primary source.

This study is also referenced in :-

The Forty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives entitled "Evaluation of Certain Food Additives and Contaminants, 1995, (section 3.2.2) as a secondary source.

The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source.

The disseminated REACH regiatration dossier for sodium nitrite also contains this study reference.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997) since only basic information is provided.

Under the conditions of this study the NOEL was determined to be 6.7 mg NO2/kg bw/day.

Further to this the Joint FAO/WHO Expert Committee on Food Additives (JECFA) established an acceptable daily nitrite intake of 0 to 0.07 mg NO2/kg bw/day by applying a safety factor of 100 to this NOEL.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic information given
Justification for type of information:
Since in the stomach the gastic juice is acidic, made up of acids and enzymes, it is suggested that in this evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In addition, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: FDA (21 CFR, Part 58)
Principles of method if other than guideline:
FDA (21 CFR, Part 58)
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Source:Taconic Farms (Gemantown, NY)
Time Held Before Studies:11 days
Average Age When Studies Began: 6 weeks
Duration of Exposure:14 weeks
Average Age at Necropsy:19 weeks (males) and 20 weeks (females)
Method of Distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights
Animals per Cage:1
Method of Animal Identification:Tail tattoo
Diet: NIH-07 open formula powdered diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water: Charcoal-filtered deionized water via amber glass bottles with stainless steel sipper tubes, available ad libitum and changed twice weekly
Cages: Solid-bottom polycarbonate
Bedding: Sani-ChipsCage
Filters: DuPont 2024 spun-bonded polyester filter (Snow Filtration Co., Cincinnati, OH)
Racks: Stainless steel
Animal Room Environment:Temperature: 72 degree +/-3 degree F
Relative humidity: 50% +/-15%
Room fluorescent light: 12 hours/dayRoom air changes: equal or more than 10/hour
Route of administration:
oral: drinking water
Vehicle:
water
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
Doses / Concentrations:0, 375, 750, 1,500, 3,000, or 5,000 ppm (equivalent to average daily doses of ca. 90, 190, 345, 750, or 990 mg/kg to males and 120, 240, 445, 840, or 1,230 mg/kg to females)
Dose / conc.:
375 ppm
Remarks:
Equivalent to 90 mg/kg for males and 120 mg/kg for females:
Basis : Nominal in water
Dose / conc.:
750 ppm
Remarks:
Equivalent to 190 mg/kg for males and 240 mg/kg for females:
Basis : Nominal in water
Dose / conc.:
1 500 ppm
Remarks:
Equivalent to 345 mg/kg for males and 445 mg/kg for females:
Basis : Nominal in water
Dose / conc.:
3 000 ppm
Remarks:
Equivalent to 750 mg/kg for males and 840 mg/kg for females:
Basis : Nominal in water
Dose / conc.:
5 000 ppm
Remarks:
Equivalent to 990 mg/kg for males and 1230 mg/kg for females:
Basis : Nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
Type and Frequency of Observation: twice daily. Core study animals were weighed and clinical findings were recorded initially, weekly, and at the end of the studies. Drinking water consumption was measured daily.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation
Necropsy: Necropsy was performed on all core study. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
Clinical Pathology: Blood for hematology and clinical chemistry was collected from the retroorbital sinus of anesthetized clinical pathology study rats on days 5 and 19 and from core study rats at the end of the study. Two blood samples each were collected from the abdominal aorta of 15 male and 15 female clinical pathology study rats on day 70 (20.00 or 22.00 hours) or 71 (09.00 hours) for hemoglobin, methemoglobin and nitrosamine concentrations; stomach contents were also collected for nitrosamine concentrations.Blood and stomach contents were collected from five males and five females at each time point.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials; erythrocyte and platelet morphologic assessments; methemoglobin concentration; reduced glutathione concentration in erythrocytes; and Heinz body count.
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.
Nitrosamine concentrations: serum and gastric nitrosamine.
Histopathology: Complete histopathology was performed on 0 and 5,000 ppm core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (female), muscle, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, skin, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. The forestomach, testis, and spleen of all remaining mice were also examined.
Other examinations:
Sperm Motility and Vaginal Cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology evaluations from male and female mice in the 0, 375, 1,500, and 5,000 ppm groups. The left cauda, epididymis, and testis were weighed. The following parameters were evaluated: spermatid heads per gram testis, spermatid heads per testis, spermatid count, motility, and concentration. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.
Statistics:
Statistical analyses were conducted about survival rate, neoplasm and non-neoplastic lesion incidences.
Details on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
Clinical signs: No clinical signs of toxicity.
Body weight and food consumption: Body weights of 990 mg/kg bw/day males were significantly less than those of the controls. Water consumption by males exposed to 1,500 ppm or greater was slightly less than that by the controls at week 13.
Mortality and time to death: All mice survived until the end of the study.
Clinical Pathology: Methaemoglobin levels were not reported.
Gross pathology incidence and severity: There were increased incidences of squamous cell hyperplasia of the forestomach in 990 mg/kg bw/day males and 1230 mg/kg bw/day females.
Organ weight changes: Relative spleen weights of 750 and 990 mg/kg bw/day males and absolute and relative heart, kidney, liver, and spleen weights of 840 and 1230 mg/kg bw/day females were greater than those of the control groups.
Histopathology: There were increased incidences of extramedullary hematopoiesis of the spleen in 750 and 990 mg/kg bw/day males and 445 mg/kg bw/day or greater females, and degeneration of the testis in 750 and 990 mg/kg bw/day males.
Sperm Motility and Vaginal Cytology: Sperm motility was decreased in 990 mg/kg bw/day males, and the estrous cycles of 445 and 1230 mg/kg bw/day females were significantly longer than in the controls.
Key result
Dose descriptor:
LOAEL
Effect level:
750 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: extramedullary haematopoiesis in the spleen, degeneration of the testis
Key result
Dose descriptor:
LOAEL
Effect level:
445 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: extramedullary haematopoiesis in the spleen
Critical effects observed:
not specified
Conclusions:
In a NTP study (2001), as a primary source, groups of male and female B6C3F1 mice (10 animals/sex/group) were exposed to 0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 90, 190, 345, 750, or 990 mg/kg mg sodium nitrite/kg bw/day in males and 0, 120, 240, 445, 840, or 1,230 mg/kg bw/day in females) in drinking water for 14 weeks.

The LOAEL were determined to be :
Males = 750 mg/kg bw/day
Females = 445 mg/kg bw/day
Executive summary:

In a NTP study (2001), as a primary source, groups of male and female B6C3F1 mice (10 animals/sex/group) were exposed to 0, 375, 750, 1,500, 3,000, or 5,000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 90, 190, 345, 750, or 990 mg/kg  mg sodium nitrite/kg bw/day in males and 0, 120, 240, 445, 840, or 1,230  mg/kg bw/day in females) in drinking water for 14 weeks.

This study is also referenced in the The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Under the conditions of the study the LOAEL were determined to be :

Males = 750 mg/kg bw/day

Females = 445 mg/kg bw/day

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic information given
Justification for type of information:
Since in the stomach the gastic juice is acidic, made up of acids and enzymes, it is suggested that in this evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In addition, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: NTP Protocol
Deviations:
not specified
Principles of method if other than guideline:
NTP Protocol
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Source: Taconic Farms (Gemantown, NY)
Time Held Before Studies:14 days (males) or 15 days (females)
Average Age When Studies Began:7 weeks
Duration of Exposure:14 weeks
Average Age at Necropsy: 20 weeks
Animals were distributed randomly into groups of approximately equal initial mean body weights
Animals per Cage: 5
Method of Animal Identification: Tail tattoo
Diet: NIH-07 open formula powdered diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water: Charcoal-filtered deionized water via amber glass bottles with stainless steel sipper tubes, available ad libitum and changed twice weekly
Cages: Solid-bottom polycarbonate
Bedding: Sani-ChipsCage
Filters: DuPont 2024 spun-bonded polyester filter (Snow Filtration Co., Cincinnati, OH)
Racks: Stainless steelAnimal Room Environment:
Temperature: 72 degree +/-3 degree F;
Relative humidity: 50% +/-15%;
Room fluorescent light: 12 hours/day;
Room air changes: Minimum 10/hour
Route of administration:
oral: drinking water
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
Doses / Concentrations:0, 750, 1500 or 3000 ppm sodium nitrite in drinking water was equivalent to average daily doses of approximately 0, 35, 70 or 130 mg/kg bw/day for males and 0, 40, 80 or 150 mg/kg bw/day for females
Dose / conc.:
750 ppm
Remarks:
Equivalent to 35 mg/kg bw/day for males and 40 mg/kg bw/day for females;
Basis: Nominal in water
Dose / conc.:
1 500 ppm
Remarks:
Equivalent to 70 mg/kg bw/day for males and 80 mg/kg bw/day for females;
Basis: Nominal in water
Dose / conc.:
3 000 ppm
Remarks:
Equivalent to 130 mg/kg bw/day for males and 150 mg/kg bw/day for females;
Basis: Nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Type and Frequency of Observation: twice daily. Core study animals were weighed and clinical findings were recorded initially, weekly, and at the end of the studies. Drinking water consumption was measured daily.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation
Necropsy: Necropsy was performed on all core study. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
Clinical Pathology: Blood for hematology and clinical chemistry was collected from the retroorbital sinus of anesthetized clinical pathology study rats on days 5 and 19 and from core study rats at the end of the study. Two blood samples each were collected from the abdominal aorta of 15 male and 15 female clinical pathology study rats on day 70 (20.00 or 22.00 hours) or 71 (09.00 hours) for hemoglobin, methemoglobin and nitrosamine concentrations; stomach contents were also collected for nitrosamine concentrations. Blood and stomach contents were collected from five males and five females at each time point.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials; erythrocyte and platelet morphologic assessments; methemoglobin concentration; reduced glutathione concentration in erythrocytes; and Heinz body count.
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.
Nitrosamine concentrations: serum and gastric nitrosamine
Histopathology: Complete histopathology was performed on 0 and 5,000 ppm core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, muscle, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, skin, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. The forestomach of 750 (males), 1,500, and 3,000 ppm animals were also examined.
Details on results:
Clinical signs: None
Mortality: Survival of exposed groups was similar to that of the controls (29/50, 38/50, 36/50 and 36/50 for males at doses of 0, 35, 70 and 130 mg/kg bw/day, respectively and 33/50, 31/50, 36/50 and 33/50 for females at 0, 40, 80 or 150 mg/kg bw/day, respectively).
Bodyweight/Food consumption: Mean body weights of 130 mg/kg bw/day males and 150 mg/kg bw/day females were less than those of the controls throughout the study. Water consumption by high dose males and females was less than that by the controls throughout the study and that by the other exposed groups was generally less after week 14.
Clinical pathology: Methaemoglobin levels were measured at two weeks and three months. At both 2 weeks and three months, methaemoglobin levels were high at night when the rats were actively feeding and drinking and low during the day when the rats were less active. Methaemoglobin levels tended to increase with increasing dosage.
Key result
Dose descriptor:
NOAEL
Effect level:
130 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
In a NTP study (2001), as a primary source, groups of male and female F344 rats (10 animals/sex/group) were exposed to 0, 750, 1,500, 3,000 ppm sodium nitrite in drinking water (equivalent to average daily doses of approximately 0, 35, 70 or 130 mg sodium nitrite/kg bw/day in males and 0, 40, 80 or 150 mg/kg bw/day in females) in drinking water for 2 years.

The NOAEL were determined to be :
Males = 130 mg/kg bw/day
Females = 150 mg/kg bw/day
Executive summary:

In a NTP study (2001), as a primary source, groups of male and female F344 rats (10 animals/sex/group) were exposed to 0, 750, 1,500, 3,000 ppm sodium nitrite in drinking water (equivalent to average daily doses of approximately 0, 35, 70 or 130 mg sodium nitrite/kg bw/day in males and 0, 40, 80 or 150 mg/kg bw/day in females) in drinking water for 2 years.

This study is also referenced in the The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Under the conditions of the study the NOAEL were determined to be :

Males = 130 mg/kg bw/day

Females = 150 mg/kg bw/day

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic information given
Justification for type of information:
Since in the stomach the gastic juice is acidic, made up of acids and enzymes, it is suggested that in this evironment it is highly unlikely that the quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites substance (s) will remain ionically bound to each other and thus are prone to dissociation in which case the released cation(s) will associate with other anions and the released anion will associate with cations. Thererfore, it is suggested that read-across data from sodium nitrite is considered appropriate in order to arrive at a conclusion in terms of any health effects that may be incurred from exposure to "nitrite" whichever cation it may be associated with.

In addition, in 1988, the US EPA, Office of Pesticides and Toxic Substances issued a Notice to producers, Formulators, Distributors and Registrants regarding quaternary ammonium compounds with regard to "Clustering" of such quaternary ammonium compounds.

Prior to this, EPA had required each quat compound to be individually coded and registered as a new chemical, even when the chemical structure of individual compounds differed only slightly in alkyl distribution and chain lengths. This procedure was continued with the new generations of quats having two, three, and four chains. As a result, EPA records showed that some 211 registered technical grade active ingredient products containing varying concentrations of quats, each coded separately on the basis of alkyl chain length and percentage carbon distribution within the chain. At this time, there are approximately eight to ten thousands (8-10,000) registered end-use formulations.

However, questions were raised regarding whether the EPA could cluster or group the quats and pick one or more representative members of each cluster to be used in toxicity studies, instead of requiring separate studies on each quat. These same questions were raised when the EPA issued its March 4, 1987 Data Call-In Notice requiring all registrants of antimicrobial active ingredients to submit subchronic and chronic toxicological data to support the continued registration of their products.

In response to these questions, EPA solicited information from industry, the public, academia, industry cooperative work groups, the state of California, and Canada. EPA then reviewed all of the assembled information along with the chemical structure of most of the quats. Based on the results of this review, EPA developed the following four groupings of currently registered quat compounds:

Group I. The alkyl or hydroxyalkyl (straight chain) substituted Quats
Group II. The non-halogenated benzyl substituted Quats (includes hydroxybenzyl, ethylbenzyl, hydroxyethybenzyl, napthylmethyl, dodecylbenzyl, and alkyl benzyl)
Group III. The di-and tri-chlorobenzyl substituted
Group IV. Quats with unusual substituents (charged heterocyclic ammonium compounds).

Fundamental to this discussion EPA determined that "X-" in all of these structures would be attributed to "any anionic species". Therefore, this would mean in terms of toxicological evaluation the coutner anion in such quaternary ammonium compounds could be regarded as; e.g halogen (Cl-, Br-, I-,), saccharinate or cyclohexylsulphamate. It is therefore suggested here that nitrite (NO2-) could also be regarded as a pertinent anion.

Since the US EPA deem that such a clustering of structures for toxicological evaluation is well founded and that the counter anion could be regarded as "any anionic species" then it would seem that to consider available toxicological data on sodium nitrite, in order to evaluate any health effects that may be incurred from exposure to the nitrite anion (NO2-), is justifiable.

Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: NTP Protocol
Deviations:
not specified
Principles of method if other than guideline:
NTP protocol
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Source:Taconic Farms (Gemantown, NY)
Time Held Before Studies:11 days
Average Age When Studies Began: 6 weeks
Duration of Exposure:14 weeks
Average Age at Necropsy:19 weeks (males) and 20 weeks (females)
Method of Distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights
Animals per Cage:1
Method of Animal Identification:Tail tattoo
Diet: NIH-07 open formula powdered diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water: Charcoal-filtered deionized water via amber glass bottles with stainless steel sipper tubes, available ad libitum and changed twice weekly
Cages: Solid-bottom polycarbonate
Bedding: Sani-ChipsCage
Filters: DuPont 2024 spun-bonded polyester filter (Snow Filtration Co., Cincinnati, OH)
Racks: Stainless steel
Animal Room Environment:Temperature: 72 degree +/-3 degree F
Relative humidity: 50% +/-15%
Room fluorescent light: 12 hours/dayRoom air changes: equal or more than 10/hour
Route of administration:
oral: drinking water
Vehicle:
water
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
Doses / Concentrations:0, 750, 1500 or 3000 ppm sodium nitrite in drinking water was equivalent to average daily doses of approximately 0, 60, 120 or 220 mg/kg bw/day for males and 0, 45, 90 or 165 mg/kg bw/day for females
Dose / conc.:
750 ppm
Remarks:
Equivalent to 60 mg/kg bw/day for males and 45 mg/kg bw/day for females;
Basis : nominal in water
Dose / conc.:
1 500 ppm
Remarks:
Equivalent to 120 mg/kg bw/day for males and 90 mg/kg bw/day for females;
Basis : nominal in water
Dose / conc.:
3 000 ppm
Remarks:
Equivalent to 220 mg/kg bw/day for males and 165 mg/kg bw/day for females;
Basis : nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Type and Frequency of Observation: twice daily. Core study animals were weighed and clinical findings were recorded initially, weekly, and at the end of the studies. Drinking water consumption was measured daily.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation
Necropsy: Necropsy was performed on all core study. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
Clinical Pathology: Blood for hematology and clinical chemistry was collected from the retroorbital sinus of anesthetized clinical pathology study rats on days 5 and 19 and from core study rats at the end of the study. Two blood samples each were collected from the abdominal aorta of 15 male and 15 female clinical pathology study rats on day 70 (20.00 or 22.00 hours) or 71 (09.00 hours) for hemoglobin, methemoglobin and nitrosamine concentrations; stomach contents were also collected for nitrosamine concentrations.Blood and stomach contents were collected from five males and five females at each time point.
Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials; erythrocyte and platelet morphologic assessments; methemoglobin concentration; reduced glutathione concentration in erythrocytes; and Heinz body count.
Other examinations:
Sperm Motility and Vaginal Cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology evaluations from male and female mice in the 0, 375, 1,500, and 5,000 ppm groups. The left cauda, epididymis, and testis were weighed. The following parameters were evaluated: spermatid heads per gram testis, spermatid heads per testis, spermatid count, motility, and concentration. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.
Statistics:
Statistical analyses were conducted about survival rate, neoplasm and non-neoplastic lesion incidences.
Details on results:
Clinical signs: none
Mortality: Survival of exposed groups was similar to that of the controls (39/50, 45/50, 42/50 and 39/50 for males at doses of 0, 60, 120 or 220 mg/kg bw/day, respectively and 40/50, 34/50, 37/50 and 41/50 for females at doses of 0, 45, 90 or 165 mg/kg bw/day, respectively).
Body weight/Food consumption: Mean body weights of 165 mg/kg bw/day females were less than those of the controls throughout the study. Exposed groups generally consumed less water than the control groups.
Clinical pathology: At 12 months, no significant increase in methaemoglobin level was observed in either sex at any dose.
Key result
Dose descriptor:
NOAEL
Effect level:
220 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
NOAEL
Effect level:
165 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
In a NTP study (2001), as a primary source, groups of male and female B6C3F1 mice (10 animals/sex/group) were exposed to 0, 750, 1500 or 3000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 60, 120 or 220 mg/kg bw/day for males and 0, 45, 90 or 165 mg/kg bw/day for females) in drinking water for 2 years.

The NOAEL were determined to be :
Males = 220 mg/kg bw/day
Females = 165 mg/kg bw/day
Executive summary:

In a NTP study (2001), as a primary source, groups of male and female B6C3F1 mice (10 animals/sex/group) were exposed to 0, 750, 1500 or 3000 ppm sodium nitrite (equivalent to average daily doses of approximately 0, 60, 120 or 220 mg/kg bw/day for males and 0, 45, 90 or 165 mg/kg bw/day for females) in drinking water for 2 years.

This study is also referenced in the The Screenimg Information DataSet (SIDS) for sodium nitrite as a secondary source and in the disseminated REACH regiatration dossier for sodium nitrite.

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Under the conditions of the study theNOAEL were determined to be :

Males = 220 mg/kg bw/day

Females = 165 mg/kg bw/day

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

As regards the repeat dose (oral) toxicity at the highest dose level the main treatment-related findings were: soft feces, body weight loss (at wk 1), lower body weight gain and food consumption, perturbation of hematological and blood biochemical parameters, distension of the cecum/colon with feces in all animals, histiocytosis, mastocytosis and sinusal hemorrhage in the mesenteric lymph node, consistent with a continued action of a mild irritant.

Justification for classification or non-classification